Tumor therapy with replication competent sindbis viral vectors

ABSTRACT

Disclosed herein are methods for treating a mammal harboring a solid tumor which expresses higher levels of High Affinity Laminin Receptors (LAMR) than normal cells of the same lineage comprising systematically administering to a mammal in need of such treatment a therapeutically effective amount of a Replication Competent (RC) Sindbis virus vector, wherein said vector encodes a suicide gene.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/030,367, filed Feb. 21, 2008, the contents of which are hereby incorporated by reference in their entirety.

The United States government has certain rights to this invention, by virtue of the funding received from grant CA100687 from the National Institutes of Health.

FIELD OF THE INVENTION

The present invention is directed to methods to treat mammals suffering from tumors and to monitor anticancer therapy using. Sindbis viral vectors and pharmaceutical formulations for use in the methods. In particular, the vectors are replication competent Sindbis Viral Vectors and the tumors are solid tumors expressing increased levels of High Affinity Laminin Receptors (LAMR) compared to normal cells of the same lineage.

BACKGROUND OF THE INVENTION

One type of gene therapy of tumors, gene-directed enzyme-prodrug therapy (GDEPT), holds considerable promise, although practical considerations limit its clinical applicability. These include the lack of acceptable noninvasive methods that are adaptable to humans for selective tumor targeting of the therapeutic genetic material. Sindbis virus is an oncolytic, alphavirus that selectively targets tumors through the 67-kDa laminin receptor (LAMR).

Gene therapy targets the genome of tumor cells as a basis for a highly selective and nontoxic anticancer therapy. To enhance selectively and specificity to the killing of cancer cells, several enzyme/prodrug systems—such as carboxylesterase/CPT-11 (1), cytosine deaminase/5-fluoro-cytosine (2), and herpes simplex virus thymidine kinase type 1 (HSVtk)/ganciclovir (GCV) (3,4)—have been developed for gene-directed enzyme-prodrug therapy (GDEPT). In this strategy, tumor cells are transduced with therapeutic genes that encode enzymes for specific conversion/activation of prodrugs, which are toxicologically inert at relatively high doses, into highly toxic metabolites for tumor killing.

In addition to a proper vector system, cancer GDEPT therapy would greatly benefit from a means to noninvasively monitor the GDEPT enzyme activity after vector treatments in vivo. Such capability could improve the Sindbis-based HSVtk/GCV GDEPT in clinical settings by providing important information to address 2 critical questions: (i) Do the vectors systemically target tumor cells and spare normal tissues? (i) Do the tumors have sufficient expression levels of the enzyme for tumor eradication by subsequent prodrug activation? In addition, monitoring during the therapy could facilitate optimizing the dose and dosing schedule of the prodrug to reduce unwanted side affects.

U.S. Pat. No. 7,306,712 discloses that vectors based on Sindbis virus, a blood-borne alphavirus transmitted through mosquito bites, infect tumor cells specifically and systemically throughout the body. The tumor specificity of Sindbis vectors may be mediated by the 67-kDa high-affinity laminin receptor (LAMR), which is over expressed in several types of human tumors. Another advantageous property of Sindbis vectors for cancer therapy is that, without carrying cytotoxic genes, they have been shown to induce apoptosis in mammalian cells. Furthermore, as Sindbis vectors are capable of expressing very high levels of their transduced suicide genes in infected tumor cells, the efficient production of the enzymes for sufficient prodrug conversion is ensured.

U.S. patent application Ser. No. 10/920,030 discloses methods and compositions for detecting cancer cells and monitoring cancer therapy using replication defective Sindbis virus vectors.

U.S. Pat. No. 7,303,798 discloses novel defective Sindbis virus vectors and their use in treating tumors in mammals.

SUMMARY OF THE INVENTION

The present inventors have unexpectedly discovered that replication competent (RC) Sindbis viral vectors have enhanced anti-tumor and cancer therapy monitoring activities when used with tumors which express higher levels of LAMR than normal cells of the same lineage. The RC Sindbis virus vectors are based on the mut-4 replication defective Sindbis virus vector disclosed in the '798 patent.

In one aspect, the present invention provides a method for treating a mammal harboring a solid tumor which expresses higher levels of High Affinity Laminin Receptors (LAMR) than normal cells of the same lineage comprising systematically administering to a mammal in need of such treatment a therapeutically effective amount of a Replication Competent (RC) Sindbis virus vector, wherein said vector encodes a suicide gene.

In another aspect, the present invention provides a method for monitoring anti-cancer therapy in a mammal harboring a solid tumor which expresses higher levels of LAMR than normal cells of the same lineage comprising administering to a mammal in need of such treatment a diagnostically effective amount of a Replication Competent (RC) Sindbis virus vector comprising a gene encoding a detectable label, and determining the amount of cancer cells in the body of said mammal, wherein the amount of cancer cells is proportional to the amount of label produced by said cancer cells and said vector encodes a suicide gene.

In a further aspect, the present invention provides a method for identifying cancer cells which expresses higher levels of LAMR than normal cells of the same lineage in the body of a mammal comprising, administering to a mammal in need of such treatment a diagnostically effective amount of a mut-4 RC Sindbis virus vector comprising a gene encoding a detectable label, and assaying for said label, wherein said cell is a cancer cell if it expresses said label and said vector encodes a suicide gene.

In a still further aspect, the present invention provides a method for determining the amount of cancer cells which expresses higher levels of LAMR than normal cells of the same lineage in the body of a mammal comprising the steps of (a) administering to a mammal in need of such treatment a diagnostically effective amount of a mut-4 RC Sindbis virus vector comprising a gene encoding a detectable label, and (b) determining the amount of said detectable label, wherein the amount of cancer cells in the body of said mammal is proportional to the amount of said label and said vector encodes a suicide gene.

In yet another aspect, the present invention provides a Replication Competent mut-4 Sindbis virus vector, wherein said vector encodes a suicide gene.

In a still further aspect, the present invention provides a pharmaceutical formulation or dosage form for administration to a mammal suffering from a solid tumor which expresses higher levels of LAMR than normal cells of the same lineage comprising a mut-4 RC Sindbis virus vector and a pharmaceutically acceptable carrier or diluent, wherein said vector further comprises a suicide gene.

These and other aspects of the present invention will be apparent to those of ordinary skill in the an in light of the present description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A-D). Various Sindbis vector systems. (A) The wild-type Sindbis virus RNA genome has two major groups of genes: non-structural genes on the 5′ side and structural genes on the 3′ side. (B) The conventional two-component replication defective (RD) system contains a replicon RNA for therapeutic gene expression, and a helper RNA to provide structural genes for vector production. (C) A replication-capable (RC) vector system with integrated structural genes. (D) a RC vector system with a suicide gene fused in-frame with the ns3 gene to achieve “controlled” vector propagation and replication in tumor cells. The ns3 gene encodes a protein which is critical for viral replication and survival.

FIG. 2(A-E). Replication-capable (RC) Sindbis virus vectors show superior tumor targeting and killing over conventional replication-defective (RD) systems. (A and B) Design of conventional RD-Sindbis/Fluc and a prototype RC-Sindbis/Fluc both carrying the firefly luciferase gene as reporter. (C) Bioluminescent imaging of tumor-bearing mice that received two consecutive daily treatments of RD- or RC-Sindbis/Fluc. The first dose was on day 0 and the last dose was on day 1. Subcutaneous BHK tumors were implanted on the right hind limb. (D) Quantitative representation of tumor signals after two consecutive treatments on day 2. (E) Tumor volume measured on day 10.

FIG. 3(A-D). A suicide gene further enhances the therapeutic efficacy of Sindbis virus vectors. (A) Design of a conventional RD vector capable of expressing HSVtk for prodrug activation. (B) Bioluminescent imaging of ES2/Fluc ovarian cancer cells that express firefly luciferase for monitoring disease progression. SCID mice were inoculated with ES2/Fluc cells on day 0. Daily treatments with RD-Sindbis/HSVtk and GCV started on day 3. The combination of Sindbis virus vectors and the prodrug ganciclovir (GCV) significantly improved anti-tumor efficacy. (C and D) Quantitative representations of the imaging data.

FIG. 4 is a map of the pSP6-R/NS3-HSVtk/Fluc-Mut4 plasmid. pSP6-R/NS3-HSVtk/Fluc-Mut4 is a Sindbis RC vector construction based on pSP6-R and Mut-4, which provides the replicase genes and structural genes respectively.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention takes advantage of the natural affinity of an alphavirus, particularly Sindbis virus, for tumor cells, in particular, for solid tumors that express higher levels of high affinity laminin receptors (alternatively referred to herein as LAMR or HALR), as compared to normal cells of the same lineage. The term “high affinity laminin receptor” or “LAMR” has its ordinary meaning in the art, i.e., the Mr 67,000 laminin receptor that can function as the receptor for Sindbis virus entry into cells (Wang et al., J. Virol. 1992, 66:4992-5001; Strauss et al., Arch. Virol. Suppl. 1994, 9:473-84).

Accordingly, the present invention provides a method for treating a mammal (e.g., human) suffering from a tumor that expresses greater levels of high affinity laminin receptor (LAMR) compared to normal cells of the same lineage. The method comprises administering to a mammal harboring such a tumor an amount of a vector effective to treat the tumor, wherein the vector has a preferential affinity for LAMR and the vector genome comprises a single component.

While not bound by any particular theory, three sets of observations may account for the remarkable anti-tumor efficiency of Sindbis vector-based therapy of the present invention. First, the LAMR can function as the receptor for Sindbis virus entry into cells of most species (Wang et al., J. Virol., 1992, 66:4992-5001; and Strauss et al., Arch. Virol. Suppl., 1994, 9:473-484). Second, it is widely recognized that expression of the LAMR is markedly elevated in many types of cancers (Menard et al., Breast Cancer Res. Treat, 1998, 52:137-145). In fact, a significant correlation has been established between the increased expression of Mr 67,000 LAMR and cancers of the breast (Menard et al., 1998, supra; Paolo Viacava et al., J. Pathol., 1997, 182:36-44; Martignone et al., J. Natl. Cancer Inst., 1993, 85:398-402), thyroid (Basolo et al., Clin. Cancer Res., 1996, 2:1777-1780), colon (San Juan et al., J Pathol., 1996, 179:376-380), prostate (Menard S et al., Breast Cancer Res. Treat, 1998, 52:137-145), stomach (de Manzoni et al., Jpn J Clin. Oncol., 1998, 28:534-537), pancreas (Pelosi et al., J Pathol., 1997, 183:62-69), ovary (Menard et al., Breast Cancer Res. Treat, 1998, 52:137-145; and van den Brule et al., Eur J Cancer, 1996, 32A:1598-1602.), melanocytes (Taraboletti et al., J Natl. Cancer Inst., 1993, 85:235-240), lung (Menard et al., Breast Cancer Res. Treat, 1998, 52:137-145), liver (Ozaki et al., Gut, 1998, 43:837-842), endometrium, and uterus (van den Brule et al., Hum Pathol, 1996, 27:1185-1191). Indeed, data on more than 4000 cases of different tumors from diverse organs studied by immunohistochemistry are all concordant with a role for HALR in invasiveness, metastasis, and tumor growth (Menard et al., Breast Cancer Res. Treat., 1998, 52:137-145). Sindbis vectors, which are naturally blood-borne, can easily travel through the circulation and specifically home to and target growing and metastatic tumors expressing increased levels of LAMR. Finally, Sindbis virus is well known to be highly apoptotic for mammalian cells (Levine et al., Nature 1993, 739-742; Jan et al. J Virol., 1999: 10296-10302; Jan et al. J Virol 2000 6425-6432). Cell death begins within a few hours of infection and by 48-96 hours virtually all infected cells are dead (Sawai et al., Mol Genet Metab. 1999, 67:36-42; Griffin et al., Ann. Rev., 1997, Microbiol. 51:565-592).

The Sindbis vectors of the present invention, do not infect normal cells to the same extent in vivo compared to tumor cells. This allows for a differential effect in vector therapy, e.g., infection by Sindbis vectors results in the death of tumor cells leading to tumor elimination without apparent deleterious effects to other tissues and organs of the treated subjects. This phenomenon may be explained by the observation that an increased number of LAMR in tumors versus normal cells leads to a high number of exposed or unoccupied receptors on tumor cells (Liotta, L. A. Cancer Research, 1986, 46:1-7; Aznavoorian et al., 1992, Molecular Aspects of Tumor Cell Invasion and Metastasis, pp. 1368-1383). For example, it has been demonstrated that breast carcinoma and colon carcinoma tissues contain a higher number of exposed (unoccupied) LAMR compared to benign lesions (Liotta et al., 1985, Exp. Cell Res., 156:117-26; Barsky et al., Breast Cancer Res. Treat., 1984, 4:181-188; Terranova et al., Proc. Natl. Acad. Sci. USA, 1983, 80: 444-448). These excess unoccupied LAMR receptors on tumor cells, which are not found in normal cells, may be available for Sindbis virus binding, infection, and induction of cell death.

The invention advantageously provides a method for treating a mammal suffering from a tumor, in which the cells of the tumor express greater levels of LAMR compared to normal cells of the same lineage. The different levels of LAMRs result in target-mediated delivery, i.e., preferential binding of vectors of the invention to tumor cells. “Greater levels” of expression generally refer herein to levels that are expressed by tumor cells (as compared to non-tumor cells) and result in such preferential binding, e.g., at least a 3-fold greater binding, preferably at least a 30-fold greater binding and, most preferably at least a 300-fold greater binding. The increased level of expression in tumor cells can be evaluated on an absolute scale, i.e., relative to any other LAMR expressing non-tumor cells described, or on a relative scale, i.e., relative to the level expressed by untransformed cells in the same lineage as the transformed cancer cells (e.g., melanocytes in the case of melanoma; hepatocytes in the case of hepatic carcinoma; ovarian endothelial cells in the case of ovarian adenocarcinoma, renal endothelial or epithelial cells in the case of renal carcinoma).

As used herein, the term “infectious”, “replication competent” or “replication capable”, when used to describe a Sindbis virus vector RNA molecule, means an RNA molecule which is self-replicating and provides for transcription in a host cell. The term “replication”, when used in conjunction with a Sindbis virus genomic RNA vector RNA molecule means production of full-length equivalents of (+)-strand RNA using (−)-strand RNA as a template.

As used herein, the term “tumor” refers to a malignant tissue comprising transformed cells that grow uncontrollably. Tumors include leukemias, lymphomas, myelomas, plasmacytomas, and the like; and solid tumors. Examples of solid tumors that can be treated according to the invention include sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, epidermoid carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, neuroglioma, and retinoblastoma. As noted above, the method of the invention depends on expression of LAMRs by cells of the tumor targeted for treatment.

The term “about” or “approximately” usually means within an acceptable error range for the type of value and method of measurement. For example, it can mean within 20%, more preferably within 10%, and most preferably still within 5% of a given value or range. Alternatively, especially in biological systems, the term “about” means within about a log (i.e., an order of magnitude) preferably within a factor of two of a given value.

The phrase “pharmaceutically acceptable”, as used in connection with compositions of the invention, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a human. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

The term “therapeutically effective” when applied to a dose or an amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a mammal in need thereof. As used herein with respect to viral vectors of the invention, the term “therapeutically effective amount/dose” refers to the amount/dose of a vector or pharmaceutical composition containing the vector that is sufficient to produce an effective anti-tumor response upon administration to a mammal.

As disclosed in Ser. No. 10/920,030 the present inventors previously discovered and devised methods for detecting tumor cells and monitoring cancer therapy. The methods are based on the natural preference of Sindbis virus to infect human cancer cells that express higher level of 37/67-KDa laminin receptor (LAMR) than normal, non-cancerous cells.

Various Sindbis viral vectors for cancer gene therapy have been designed based on the RNA genome of wild-type virus (FIG. 1A). The wild-type genome contains two major parts. The first part, located on the 5′ side of the genome, carries all of the non-structural genes (ns1, ns2, ns3, and ns4) and a packaging signal in the ns1 region. The expression of the ns1-4 genes leads to synthesis of the non-structural proteins nsp 1-4 that form the viral replicase. The replicase is necessary for expression of the structural genes in the second part of the genome at 3′ side for virus formation. In order to do so, the replicase specifically recognizes a short stretch of sequences between the non-structural and structural genes, called the sub-genomic promoter (P_(SG)). One structural protein, the capsid protein, specifically recognizes the packaging signal at the list region and picks it up to form viral particles with the rest of the structural proteins.

FIG. 1B depicts the conventional design of a replication defective Sindbis viral vector that is capable of efficient delivery of therapeutic genes while being unable to propagate and generate more vector particles. This particular system has two components. Replicon RNA contains all of the non-structural genes, while the structural genes are replaced with the therapeutic gene to be delivered. In order to generate vector particles, the structural genes are provided in trans using a helper RNA, the second component of this system. Unlike replicon RNA, the helper RNA contains the non-structural genes and lacks the packaging signal. Therefore, the produced vector particles only carry replicon RNA with the gene of interest.

One major advantage of such conventional dual-component vector systems is that the produced vector particle is replication-defective (RD) and is safer for clinical use. However, this advantage may become a significant drawback for cancer genes therapy. The goal of cancer gene therapy is to infect the majority of tumor cells and deliver the therapeutic genes for tumor detection or eradication. To achieve this goal using a replication-defective system may require a repetitive treatment regime and high doses of vectors. In some cases, such high doses may not be easily obtained using a replication defective system.

On the other hand, a Sindbis vector system that is capable of “controlled” replication and propagation is of great interest for cancer gene therapy. Such a replication-capable (RC) system should comprise a single-component to ensure efficient propagation of the vector in the tumor. That is, the system does not require a helper component for replication. One major benefit of such a system is that fewer treatments would be required and a lower dose should be sufficient to achieve successful therapeutic outcomes while retaining the same tumor targeting capability as RD vectors. Preferably, a safety mechanism is incorporated in the vector to ensure that the vector is eliminated in order to prevent unwanted toxicity, if any, associated with the propagation of the vector.

The present invention provides a replication-capable (RC) Sindbis viral vector (FIG. 1C) which was tested to see if a single-component system performed better than the conventional dual-component system. Instead of using a separate helper RNA vector to carry the structural genes, the structural genes are directly inserted into a conventional replicon vector, along with a dedicated sub-genomic promoter following the therapeutic gene. The dedicated second promoter guarantees that the efficient expression of the structural genes for high-level vector production occurs. A simple dual-promoter RC system was first developed in order to study the function of the sub-genomic promoter in mammalian cells (5,6). This type of RC system was later used to deliver antigen encoding genes for vaccination purposes (7-9). Since Sindbis virus infects mosquito cells, a simple RC system was also used to study Sindbis virus spreading in mosquitoes (10). In light of the present inventors' discovery that RD Sindbis vectors can target tumors in living animals, a single-component RC Sindbis vector has been used to detect/treat tumors in mouse tumor models (11). However, instead of using a dedicated sub-genomic promoter, the prior art RC system used a cleavable component to release the reporter protein from the structural proteins and, therefore, significantly reduced the titer of vector production (12).

FIG. 2 shows the results of an experiment comparing the anti-tumor activity of replication defective (RD, FIG. 2A) and replication competent (RC, FIG. 2B) Sindbis virus vectors. In this set of experiments, a subcutaneously induced BHK tumor model (on the right hind limb of SCID mice) was used to test and compare these two vector systems. In order to evaluate specific tumor targeting and elimination in this model, both vector systems carry a firefly luciferase gene as a reporter. The bioluminescent signals generated in the tumors can be easily detected and analyzed using the IVIS™ imaging system. Mice received only two consecutive treatments of RD or RC vectors via systemic (intravenous) injections on a day 0 and day 1. No further treatment was administrated. FIG. 2C depicts the imaging result on day 2 and FIG. 2D quantitatively shows the level of bioluminescent signals in tumors that directly reflects vector infection level. The data indicate that the RC vector system has about a 30 fold increase in infectivity compared to the conventional RD vector system. Higher infectivity is also reflected in enhanced tumor killing as evidenced by tumor size reduction on day 10 (FIG. 2E).

These data provide proof-of-concept results in support of the use of a RC Sindbis vector system for cancer gene therapy. The capability of the RC vector to propagate and spread to the tumor dramatically enhances the ability of Sindbis vectors to target and kill cancer cells. The data also show that the same level of tumor detection can be achieved using a lower range of effective doses of the RC vector compared to conventional RD vectors. In the example depicted in FIG. 2, 10⁶ RD or RC vector particles were intravenously administered into mice. The signals from tumors treated with the RC vectors were about 30 fold higher than the RD signals. Therefore, the same imaging intensity in tumors was achieved using a lower dose (10⁴ to 10⁵) of RC vectors. Interestingly, no toxicity was associated with this prototype RC vector administered at 10⁶ level and no bioluminescent signals, except in residual tumors, were observed in animals up to 20 days after vector injections, suggesting that using the same level of RC vector, as high as 10⁶ per dose, should not cause side effects. The higher dose is useful not only for detecting small lesions but also for better tumor killing and therapeutic effects. Since humans are about 1000 times the body weight of mice, the expected range of effective amounts for humans will range between about 10⁷ and about 10¹⁰ particles per dose. The lower range (10⁷-10⁸) is sufficient to detect tumor masses and higher range (10⁸-10¹⁰) are better for tumor eradication.

For use in the present invention, the RC Sindbis virus vectors can be produced as described in U.S. Pat. Nos. 7,306,712 and 7,303,798 and in the example below. This involves the described in vitro transcription/electroporation method.

However, as mentioned above, a safety mechanism significantly reduces the risk, if any, of toxicity by controlling the propagation of the RC vector system. In a preferred embodiment, a “suicide gene” is incorporated into one of the Sindbis virus non-structural genes that are essential for viral propagation and survival. FIG. 1C depicts the concept of such a design. A suicide gene, which encodes an enzyme capable of activating inert prodrugs into cytotoxic metabolites, is fused in frame with the ns3 gene to ensure co-expression with non-structural protein 3, an essential component of viral replicase. A particular region of the ns3 gene has been shown to be suitable for fusion without compromising the function of nsp3. Therefore, by such design the RC vector is genetically tagged with this safety mechanism which can shut-off vector propagation by killing the vector producing cell during or after the treatment regime.

In addition to serving as a safety feature, the fused suicide gene provides another advantage. The tumor cells that are selectively infected by the vector are more susceptible and sensitive to the prodrug treatment, since they would not only face the killing imposed by Sindbis infection, but also are exposed to toxic metabolites as a result of prodrug activation. In this regard, it has been discovered that activated toxic metabolites can passively diffuse to neighboring uninfected tumor cells to further enhance tumor killing. This is called a “bystander effect”. The bystander effect plays an important role in the eradication of surrounding untransduced (uninfected) tumor cells. This is caused by transmission of the activated prodrug from the transduced tumor cells (which may be only a small fraction of total tumor mass) to uninfected tumor cells. In the HSVtk/GCV system, the activated GCV is not membrane permeable because of its highly charged phosphate groups. However, it can be transferred to uninfected cells via the gap junctions or through the exchange of apoptotic vesicles that kill the surrounding untransduced tumor cells (14).

Several suicide genes and their appropriate prodrugs are available and suitable for use with the Sindbis virus vector in this embodiment. For example, as disclosed herein, a conventional Sindbis virus vector carrying a thymidine kinase gene isolated from herpes simplex virus (HSVtk) significantly enhanced tumor killing (FIG. 3A) (Horsburgh B C et al, Recurrent acyclovir-resistant herpes simplex in an immunocompromised patient; can strain differences compensate for loss of thymidine kinase in pathogenesis? J. Infect. Dis., 178 (3), 618-625, 1998). A specific prodrug, ganciclovir (GCV), has been developed to target HSVtk, and has been clinically approved for treatment of cytomegalovirus and herpes simplex virus infection in humans. (Ganciclovir, GCV, is marketed under the trade name CYTOVENE™ by Roche Laboratories Inc.)

Additional examples of suicide genes are thymidine kinase of Varicella Zoster virus (VZV-tk) (disclosed in Lacey S F et al, Analysis of mutations in the thymidine kinase genes of drug-resistant varicella-zoster virus populations using the polymerase chain reaction, J. Gen. Virol. 72 (PT 3), 623-630, 1991) and the bacterial gene cytosine deaminase (Perna N T et al, Genome sequence of enterohaemorrhagic Escherichia coli O157:H7, Nature 409 (6819), 529-533, 2001).

The prodrugs useful in the methods of the present invention are any that can be converted to a toxic product, i.e., toxic to tumor cells. A preferred prodrug is ganciclovir, which is converted in vivo to a toxic compound by HSV-tk (Chen et al, Cancer Res. 1996, 56: 3758-3762). Other representative examples of prodrugs include acyclovir, FIAU [1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil] (FIALURIDINE™, Moravek Biochemicals and Radiochemicals), 6-methoxypurine arabinoside (converted by VZV-tk), and 5-fluorocytosine (converted by cytosine deaminise) (5-fluorocytosine, Roche).

Prodrugs, may be readily administered to patients by physicians having ordinary skill in the art. Using methods known in the field, such physicians would also be able to determine the most appropriate dose and route for the administration of the prodrug. For example, ganciclovir is preferably administered systemically (e.g. orally or parenterally) in a dose of about 1-20 mg/day/kg body weight; acyclovir is administered in a dose of about 1-100 mg/day/kg body weight, and FIAU is administered in a dose of about 1-50 mg/day/kg body weight.

In the example below, SCID mice were intraperitoneally implanted with ES2 human ovarian cancer cells. In order to track and monitor disease progression, the ES2 cells were genetically engineered to express a firefly luciferase gene for bioluminescent imaging. Therefore, the bioluminescent signal intensity is proportional to the tumor load in these animals (FIG. 3B). Tumor-bearing mice were either mock treated or received daily treatments with a conventional RD-Sindbis/HSVtk vector. Some mice also received daily GCV treatments to determine if the prodrug enhanced tumor killing in conjunction with Sindbis vector treatment. Quantitative analysis indicated that, without HSVtk expression, unactivated GCV confers no therapeutic effect on tumor loading (FIG. 3C). On the contrary, GCV dramatically enhanced the Sindbis/HSVtk vector treatments and suppressed tumor growth (FIG. 3D).

In an alternate embodiment, the vectors of the present invention can be used to detect cancer cells and monitor anti-cancer therapy. Previously, the present inventors used an optical bioluminescence imaging system and RD Sindbis virus vectors to detect tumor-specific targeting of Sindbis virus vectors in small animals (15, 16 and Ser. No. 10/920,030). The advantages of bioluminescent imaging include short imaging time, low costs, and ease of use. However, optical imaging methods suffer from very substantial attenuation of the light, signal and, thus, are not amenable to applications in large animals and in patients. Recent advances in optical and radionuclide imaging technology provide several methods for non-invasive monitoring of marker gene expression in living animals. On the other hand, radionuclide imaging methods such as g-camera, SPECT, and PET have excellent depth sensitivity and can detect accumulation of gene expression within the transfected tumors anywhere in the body and on the basis of gene expression imaging (17,18,19). A major advantage of PET is the ability to generate quantitative high spatial resolution, 3-dimensional images. When combined with other forms of tomographic imaging, such as CT or MRI, fusion images of functional and anatomic data provides more detailed in situ information of marker genes' expression and localization.

As disclosed in copending Ser. No. 10/920,030, the present inventors have discovered that imaging can be translated into photon counts produced by the detectable label delivered to cancer cells and that these are proportional to the amount of tumor cells that remain alive. Therefore, the present invention can be used to monitor anti-cancer therapy as follows. Patients can be administered a diagnostically-effective amount of the RC Sindbis vector of the present invention comprising a detectable label before the onset of treatment, and this value can be compared to one obtained upon administration of a diagnostically effective amount of a Sindbis virus comprising a detectable label after therapy has been completed. In this way, it is possible to determine the extent of tumor kill. Since only living tumor cells would contain the label, therapy would continue only until a minimal amount of label is detected.

Since Sindbis virus vectors are gene transfer vectors, the cancer cells are labeled using genetic markers incorporated into the RC Sindbis virus vectors. In this embodiment, the genes useful for live tumor monitoring or labeling include but are not limited to the Green Fluorescence Protein (GFP) gene, [Cormack, B. P. et al. (1966) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173:33-38] the Firefly luciferase (Fluc) gene, [de Wet, J. R., et al. (1987) Firefly luciferase gene: structure and expression in mammalian cells Mol. Cell Biol. 7 (2), 725-737], the Renilla luciferase (Rluc) gene [Lorenz, W. W. et al. (1991) Isolation and expression of a cDNA encoding Renilla reinformis luciferase, Proc. Natl. Acad. Sci. U.S.A. 88 (10), 4438-4442] and the dopamine-2 receptor (D₂R) gene. The use of the D₂R gene as a reporter gene in living animals is disclosed in MacLaren et al. (Gene Therapy 6:785-791 (1999)) and Yaghoubi et al. (Gene Therapy 8:1072-1080 (2001)) These genes can be incorporated into the Sindbis virus vectors of the present invention using techniques well known to those of ordinary skill in the art, as described in Bredenbeek P. J. et al. (1993) (Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs, J. Virol.; 67(11):6439-46.)

Cells expressing the genetic markers of the present invention can be identified as follows: for HSV-tk gene, the subject can be administered radiolabeled 9-[(4[¹⁸F]fluro-3-hydroxymethylbutyl)guanine (FHBG), administered intravenously, about 6000 μCi/Kg body weight of the recipient, (commercially available from PET Imaging Science Center, U. of South California). Expression of HSV-tk activity in tumor cells results in the accumulation of radiolabeled FHBG and can be monitored by Positron Emission Tomography (PET). In vivo GFP expressing tumor cells can be monitored by fluorescence microscopic examination of tissue sections. Tissue sections of Fluc or Rluc expressing tumor cells can be monitored by Cooled Charge-Coupled Device (CCD) cameras in viva (commercially available from Xenogen Corp., Alamenda, Calif.). D₂R activity can be identified by administering 3-(2-[¹⁸F]fluoroethyl)spiperone ([¹⁸F]FESP) and monitored by PET.

A subject to whom the diagnostic compound of the present invention has been administered as an effective diagnostic monitor for a disease or disorder is preferably a human, but can be any animal, including a laboratory animal in the context of a clinical trial or screening or activity experiment. Thus, as can be readily appreciated by those of ordinary skill in the art, the methods and compositions of the present invention are particularly suited to administration to any animal, particularly a mammal, and including, but by no means limited to, domestic animals, such as feline or canine subjects, farm animals, such as but not limited to bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avian species, such as chickens, turkeys, songbirds, etc., i.e., for veterinary medical use.

In summary, the single-component RC Sindbis vector system of the present invention dramatically enhances the tumor targeting, monitoring and killing capability of replication-capable Sindbis vectors, and incorporation of suicide genes provides an additional layer of protection to achieve “controlled” propagation in tumors and enhances tumor cell killing by RC Sindbis virus vectors.

In a preferred embodiment, the RC vectors are derived from the RD mut-4 vector disclosed in U.S. Pat. No. 7,303,798. The mut-4 vector is similar to the SinRep5 system (Invitrogen Corp.), except for changes in the amino acid sequences in the E2 protein. Since this protein is directly involved in vector binding and targeting to tumor cells, it is expected that RC vectors derived from the mut-4 vector will have the same improved binding capability as the RD vectors.

The construction of the RC mut-4 vector containing the HSV-tk gene is shown in Paper Example 1 below.

The present invention also provides pharmaceutical formulations or dosage forms for administration to mammals.

When formulated in a pharmaceutical composition, the vectors of the present invention can be admixed with a pharmaceutically acceptable carrier or excipient. The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are “generally regarded as safe”, e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicles with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

The preferred route of administration of the vectors of the present invention, for treatment and monitoring, is parenteral and most preferably systemic. This includes, but is not limited to intravenous, intraperitoneal, intra-arteriole, intra-muscular, intradermal, subcutaneous, intranasal and oral. These routes of administration will permit homing of the vector to tumor cells wherein, only Sindbis virus is a blood-borne virus. Therefore, gene therapy vectors based on this virus have an advantage over other viral vectors that are not adapted to travel in the bloodstream. This property is largely responsible for the observation that systemic administration of Sindbis viral vectors by i.p. or i.v. injections, target and infect only tumors expressing greater amounts of LAMR than normal cells of the same lineage growing s.c., i.p., intrapancreatically, or in the lungs. Thus, the blood-borne nature of Sindbis viral vectors provides them with the capacity to treat malignancies and monitor cancer therapy.

The present invention is described below in examples which are intended to further describe the invention without limiting the scope therapy.

Materials and Methods

Nectar Construction

The prototype RC-Sindbis/Fluc vector was constructed using the pSinRep5/Fluc plasmid as a backbone. In order to make the vector replication competent, a DNA segment containing a sub-genomic promoter and the Sindbis viral structural genes was excised from the ptRNA-DHBB plasmid (Invitrogen Corp., Carlsbad, Calif.) using NsiI and BamI enzymes and then inserted into pSinRep5/Fluc at the StuI site. Therefore the constructed pSinRep5/Fluc-tBB plasmid has two independent sub-genomic promoters to drive expression of firefly luciferase and Sindbis viral structural proteins.

For construction of a RC vector with a suicide gene in the HSVtk gene was fused in-frame with the Ns3 gene at the SpeI site in pSinRep5/Fluc-tBB. The HSVtk gene (from the pORF-HSVtk plasmid, Invivogen, San Diego, Calif.) was inserted at this site to generate the pSinRep5-nsp3-HSVtk/Fluc-tBB plasmid.

Vector Preparation

The RC-Sindbis/Fluc vector was prepared using an in vitro transcription/electroporation method as described U.S. Pat. Nos. 7,306,712 and 7,303,798. The plasmid pSinRep5/Fluc-tBB was linearized using the NotI restriction enzyme. The linearized plasmid DNA was then used as template for in vitro transcription. The in vitro transcription was done in a total volume of 20 μL using a commercially available SP6 in vitro transcription kit (Ambion Inc., Austin, Tex.). Transcribed RNA (20 μL) was then electroporated into 6×10⁶ BHK cells and cultured at 37° C. in a 10 cm dish containing 10 mL of αMEM (Invitrogen Corp.) with 10% FCS. The next day, the culture media was replaced with 9 mL of OptiMEM (Invitrogen). The OptiMEM was then harvested and stored at −80° C.

In Vivo Imaging

BHK tumors were induced in female SCID mice (Taconic, Germantown, N.Y.) by subcutaneous injection of 2 million BHK cells. Ten days later, on day 0, mice were split into two groups. One group of five mice received the first intravenous injection of 10⁶ particles of RD-Sindbis/Fluc, and the other group received the first i.v. injection of 10⁶ particles of RC-Sindbis/Fluc. The next day (day 1), both groups received a second dose (10⁶) of i.v. treatments. Twenty-four hours later (day 2), tumor luminescence signals were measured using the IVIS® spectrum imaging system (Caliper LifeSciences, Hopkinton, Mass.) and tumor specific signals were analyzed using Living Image 3.0 software. Five minutes before imaging, 0.3 mL of 15 mg/mL D-luciferin (Promega, Madison, Wis.) was i.p. injected in order to generate bioluminescent signals. Tumor sizes were measured using calipers and volumes were calculated using the formula: 4π/3×length×width×height.

An ES-2/Fluc ovarian cancer model was used to test if the HSVtk suicide gene enhanced the therapeutic effects of Sindbis vectors. The prodrug GCV (CYTOVENE-IV®, The Roche Laboratories Inc.) enhanced the killing of Sindbis/tk-infected ES-2/Fluc cells in vivo, as determined by the IVIS® system, which is capable of non-invasive detection of bioluminescent signal generated by ES-2/Fluc tumors. SCID mice were inoculated with ES-2/Fluc on day 0. Daily GDEPT treatments involving i.p. injections of RD-Sindbis/tk and GCV (25 mg/Kg of body weight) were started on day 3. The Sindbis/tk −GCV group (n=5) received Sindbis/tk treatments but no GCV. The Sindbis/tk +GCV group (n=5) received both Sindbis/tk and GCV treatments. The Control group (n=5) was neither treated with Sindbis/tk nor GCV. The Control +GCV group (n=5) received no Sindbis/tk but was treated with GCV. Disease progression was monitored and the whole body photon counts were determined using the IVIS® system on days 1, 3, 6, 8, 12 and 14. Representative images of each treatment group are shown in FIG. 2.

In FIG. 2, tumor luciferase signals were higher and tumor volumes were lower using the RC vector compared to the RD vector. The average tumor luciferase signal in RC-treated mice was 43,970,000 photons, which was much higher than the average of 1,776,000 photons in RD-treated animals. The average tumor size of RC-treated tumors was 410 mm3 and for RD-treated tumor the average size was 1609 mm3.

Paper Example 1

The plasmid pSP6-R/NS3-HSVtk/Fluc-Mut4 enclodes a Sindbis virus RC vector construction based on pSP6-R and Mut-4, which provide replicase genes and structural genes respectively. Its sequence is set forth in Appendix A and a map of the plasmid is shown in FIG. 4. In an alternate embodiment, the promoter is T7 (Ambun, Austin, Tex.??) Its construction is described as follows.

A DNA segment containing a sub-genomic promoter and the Sindbis viral structural genes is excised from the pSP6-Mut4 plasmid (disclosed in U.S. Pat. No. 7,303,798) using NsiI and BamHI enzymes and then inserted into pSP6-R/Fluc at the PmlI site. In addition, the HSVtk gene fragment (from the pORF-HSVtk plasmid (Invivogen, San Diego, Calif.) is inserted at the SpeI site in the ns3 region on pSP6-R/Fluc-tBB to generate the pSP6-R/nsp3-HSVtk/Fluc-tBB plasmid. A map showing the pSP6-R/nsp3-HSVtk/Fluc-tBB plasmid is shown in FIG. 4 and its sequence is set forth in Appendix A below.

In FIG. 4, the location of the genes is set forth below:

Sindbis non-structural genes locations (bp):

NS1  60-1979 (SEQ. ID NO.: 2) NS2 1980-4100 (SEQ. ID NO.: 3) NS3 4101-5261 and 6399-6878 (SEQ. ID NO.: 4) NS4 6879-8729 (SEQ. ID NO.: 5) First Psg 8722-8734 (SEQ. ID NO.: 6) Second Psg 10766-10789 (SEQ. ID NO. 7)  Structural Genes: 10833-14567 (SEQ. ID NO.: 8) 

In addition, the vector may also comprise a suicide gene, such as the thymidine kinase (TK) gene located within the NS3 gene (Nucleotide 5262-6398).

REFERENCES

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APPENDIX A pSP6-R/nsp3-HSVtk/Fluc-Mut4 1 ATTGACGGCG TAGTACACAC TATTGAATCA AACAGCCGAC CAATTGCACT TAACTGCCGC ATCATGTGTG ATAACTTAGT TTGTCGGCTG GTTAACGTGA 51 ACCATCACAA TGGAGAAGCC AGTAGTAAAC GTAGACGTAG ACCCCCAGAG TGGTAGTGTT ACCTCTTCGG TCAGCATGTG CATCTGCATC TGGGGGTCTC 101 TCCGTTTGTC GTGCAACTGC AAAAAAGCTT CCCGCAATTT GAGGTAGTAG AGGCAAACAG CACGTTGACG TTTTTTCGAA GGGCGTTAAA CTCCATCATC 151 CACAGCAGGT CACTCCAAAT GACCATGCTA ATGCCAGAGC ATTTTCGCAT GTGTCGTCCA GTGAGGTTTA CTGGTACGAT TACGGTCTCG TAAAAGCGTA 201 CTGGCCAGTA AACTAATCGA GCTGGAGGTT CCTACCACAG CGACGATCTT GACCGGTCAT TTGATTAGCT CGACCTCCAA GGATGGTGTC GCTGCTAGAA 251 GGACATAGGC AGCGCACCGG CTCGTAGAAT GTTTTCCGAG CACCAGTATC CCTGTATCCG TCGCGTGGCC GAGCATCTTA CAAAAGGCTC GTGGTCATAG 301 ATTGTGTCTG CCCCATGCGT AGTCCAGAAG ACCCGGACCG CATGATGAAA TAACACAGAC GGGGTACGCA TCAGGTCTTC TGGGCCTGGC GTACTACTTT 351 TATGCCAGTA AACTGGCGGA AAAAGCGTGC AAGATTACAA ACAAGAACTT ATACGGTCAT TTGACCGCCT TTTTCGCACG TTCTAATGTT TGTTCTTGAA 401 GCATGAGAAG ATTAAGGATC TCCGGACCGT ACTTGATACG CCGGATGCTG CGTACTCTTC TAATTCCTAG AGGCCTGGCA TGAACTATGC GGCCTACGAC 451 AAACACCATC GCTCTGCTTT CACAACGATG TTACCTGCAA CATGCGTGCC TTTGTGGTAG CGAGACGAAA GTGTTGCTAC AATGGACGTT GTACGCACGG 501 GAATATTCCG TCATGCAGGA CGTGTATATC AACGCTCCCG GAACTATCTA CTTATAAGGC AGTACGTCCT GCACATATAG TTGCGAGGGC CTTGATAGAT 551 TCATCAGGCT ATGAAAGGCG TGCGGACCCT GTACTGGATT GGCTTCGACA AGTAGTCCGA TACTTTCCGC ACGCCTGGGA CATGACCTAA CCGAAGCTGT 601 CCACCCAGTT CATGTTCTCG GCTATGGCAG GTTCGTACCC TGCGTACAAC GGTGGGTCAA GTACAAGAGC CGATACCGTC CAAGCATGGG ACGCATGTTG 651 ACCAACTGGG CCGACGAGAA AGTCCTTGAA GCGCGTAACA TCGGACTTTG TGGTTGACCC GGCTGCTCTT TCAGGAACTT CGCGCATTGT AGCCTGAAAC 701 CAGCACAAAG CTGAGTGAAG GTAGGACAGG AAAATTGTCG ATAATGAGGA GTCGTGTTTC GACTCACTTC CATCCTGTCC TTTTAACAGC TATTACTCCT 751 AGAAGGAGTT GAAGCCCGGG TCGCGGGTTT ATTTCTCCGT AGGATCGACA TCTTCCTCAA CTTCGGGCCC AGCGCCCAAA TAAAGAGGCA TCCTAGCTGT 801 CTTTATCCAG AACACAGAGC CAGCTTGCAG AGCTGGCATC TTCCATCGGT GAAATAGGTC TTGTGTCTCG GTCGAACGTC TCGACCGTAG AAGGTAGCCA 851 GTTCCACTTG AATGGAAAGC AGTCGTACAC TTGCCGCTGT GATACAGTGG CAAGGTGAAC TTACCTTTCG TCAGCATGTG AACGGCGACA CTATGTCACC 901 TGAGTTGCGA AGGCTACGTA GTGAAGAAAA TCACCATCAG TCCCGGGATC ACTCAACGCT TCCGATGCAT CACTTCTTTT AGTGGTAGTC AGGGCCCTAG 951 ACGGGAGAAA CCGTGGGATA CGCGGTTACA CACAATAGCG AGGGCTTCTT TGCCCTCTTT GGCACCCTAT GCGCCAATGT GTGTTATCGC TCCCGAAGAA 1001 GCTATGCAAA GTTACTGACA CAGTAAAAGG AGAACGGGTA TCGTTCCCTG CGATACGTTT CAATGACTGT GTCATTTTCC TCTTGCCCAT AGCAAGGGAC 1051 TGTGCACGTA CATCCCGGCC ACCATATGCG ATCAGATGAC TGGTATAATG ACACGTGCAT GTAGGGCCGG TGGTATACGC TAGTCTACTG ACCATATTAC 1101 GCCACGGATA TATCACCTGA CGATGCACAA AAACTTCTGG TTGGGCTCAA CGGTGCCTAT ATAGTGGACT GCTACGTGTT TTTGAAGACC AACCCGAGTT 1151 CCAGCGAATT GTCATTAACG GTAGGACTAA CAGGAACACC AACACCATGC GGTCGCTTAA CAGTAATTGC CATCCTGATT GTCCTTGTGG TTGTGGTACG 1201 AAAATTACCT TCTGCCGATC ATAGCACAAG GGTTCAGCAA ATGGGCTAAG TTTTAATGGA AGACGGCTAG TATCGTGTTC CCAAGTCGTT TACCCGATTC 1251 GAGCGCAAGG ATGATCTTGA TAACGAGAAA ATGCTGGGTA CTAGAGAACG CTCGCGTTCC TACTAGAACT ATTGCTCTTT TACGACCCAT GATCTCTTGC 1301 CAAGCTTACG TATGGCTGCT TGTGGGCGTT TCGCACTAAG AAAGTACATT GTTCGAATGC ATACCGACGA ACACCCGCAA AGCGTGATTC TTTCATGTAA 1351 CGTTTTATCG CCCACCTGGA ACGCAGACCA TCGTAAAAGT CCCAGCCTCT GCAAAATAGC GGGTGGACCT TGCGTCTGGT AGCATTTTCA GGGTCGGAGA 1401 TTTAGCGCTT TTCCCATGTC GTCCGTATGG ACGACCTCTT TGCCCATGTC AAATCGCGAA AAGGGTACAG CAGGCATACC TGCTGGAGAA ACGGGTACAG 1451 GCTGAGGCAG AAATTGAAAC TGGCATTGCA ACCAAAGAAG GAGGAAAAAC CGACTCCGTC TTTAACTTTG ACCGTAACGT TGGTTTCTTC CTCCTTTTTG 1501 TGCTGCAGGT CTCGGAGGAA TTAGTCATGG AGGCCAAGGC TGCTTTTGAG ACGACGTCCA GAGCCTCCTT AATCAGTACC TCCGGTTCCG ACGAAAACTC 1551 GATGCTCAGG AGGAAGCCAG AGCGGAGAAG CTCCGAGAAG CACTTCCACC CTACGAGTCC TCCTTCGGTC TCGCCTCTTC GAGGCTCTTC GTGAAGGTGG 1601 ATTAGTGGCA GACAAAGGCA TCGAGGCAGC CGCAGAAGTT GTCTGCGAAG TAATCACCGT CTGTTTCCGT AGCTCCGTCG GCGTCTTCAA CAGACGCTTC 1651 TGGAGGGGCT CCAGGCGGAC ATCGGAGCAG CATTAGTTGA AACCCCGCGC ACCTCCCCGA GGTCCGCCTG TAGCCTCGTC GTAATCAACT TTGGGGCGCG 1701 GGTCACGTAA GGATAATACC TCAACCAAAT GACCGTATGA TCGGACAGTA CCAGTGCATT CCTATTATGG AGTTCGTTTA CTGGCATACT AGCCTGTCAT 1751 TATCGTTGTC TCGCCAAACT CTGTGCTGAA GAATGCCAAA CTCGCACCAG ATAGCAACAG AGCGGTTTGA GACACGACTT CTTACGGTTT GAGCGTGGTC 1801 CGCACCCGCT AGCAGATCAG GTTAAGATCA TAACACACTC CGGAAGATCA GCGTGGGCGA TCGTCTAGTC CAATTCTAGT ATTGTGTGAG GCCTTCTAGT 1851 GGAAGGTACG CGGTCGAACC ATACGACGCT AAAGTACTGA TGCCAGCAGG CCTTCCATGC GCCAGCTTGG TATGCTGCGA TTTCATGACT ACGGTCGTCC 1901 AGGTGCCGTA CCATGGCCAG AATTCCTAGC ACTGAGTGAG AGCGCCACGT TCCACGGCAT GGTACCGGTC TTAAGGATCG TGACTCACTC TCGCGGTGCA 1951 TAGTGTACAA CGAAAGAGAG TTTGTGAACC GCAAACTATA CCACATTOCC ATCACATGTT GCTTTCTCTC AAACACTTGG CGTTTGATAT GGTGTAACGG 2001 ATGCATGGCC CCGCCAAGAA TACAGAAGAG GAGCAGTACA AGGTTACAAA TACGTACCGG GGCGGTTCTT ATGTCTTCTC CTCGTCATGT TCCAATGTTT 2051 GGCAGAGCTT GCAGAAACAG AGTACGTGTT TGACGTGGAC AAGAAGCGTT CCGTCTCGAA CGTCTTTGTC TCATGCACAA ACTGCACCTG TTCTTCGCAA 2101 GCGTTAAGAA GGAAGAAGCC TCAGGTCTGG TCCTCTCGGG AGAACTGACC CGCAATTCTT CCTTCTTCGG AGTCCAGACC AGGAGAGCCC TCTTGACTGG 2151 AACCCTCCCT ATCATGAGCT AGCTCTGGAG GGACTGAAGA CCCGACCTGC TTGGGAGGGA TAGTACTCGA TCGAGACCTC CCTGACTTCT GGGCTGGACG 2201 GGTCCCGTAC AAGGTCGAAA CAATAGGAGT GATAGGCACA CCGGGGTCGG CCAGGGCATG TTCCAGCTTT GTTATCCTCA CTATCCGTGT GGCCCCAGCC 2251 GCAAGTCAGC TATTATCAAG TCAACTGTCA CGGCACGAGA TCTTGTTACC CGTTCAGTCG ATAATAGTTC AGTTGACAGT GCCGTGCTCT AGAACAATGG 2301 AGCGGAAAGA AAGAAAATTG TCGCGAAATT GAGGCCGACG TGCTAAGACT TCGCCTTTCT TTCTTTTAAC AGCGCTTTAA CTCCGGCTGC ACGATTCTGA 2351 GAGGGGTATG CAGATTACGT CGAAGACAGT AGATTCGGTT ATGCTCAACG CTCCCCATAC GTCTAATGCA GCTTCTGTCA TCTAAGCCAA TACGAGTTGC 2401 GATGCCACAA AGCCGTAGAA GTGCTGTACG TTGACGAAGC GTTCGCGTGC CTACGGTGTT TCGGCATCTT CACGACATGC AACTGCTTCG CAAGCGCACG 2451 CACGCAGGAG CACTACTTGC CTTGATTGCT ATCGTCAGGC CCCGCAAGAA GTGCGTCCTC GTGATGAACG GAACTAACGA TAGCAGTCCG GGGCGTTCTT 2501 GGTAGTACTA TGCGGAGACC CCATGCAATG CGGATTCTTC AACATGATGC CCATCATGAT ACGCCTCTGG GGTACGTTAC GCCTAAGAAG TTGTACTACG 2551 AACTAAAGGT ACATTTCAAT CACCCTGAAA AAGACATATG CACCAAGACA TTGATTTCCA TGTAAAGTTA GTGGGACTTT TTCTGTATAC GTGGTTCTGT 2601 TTCTACAAGT ATATCTCCCG GCGTTGCACA CAGCCAGTTA CAGCTATTGT AAGATGTTCA TATAGAGGGC CGCAACGTGT GTCGGTCAAT GTCGATAACA 2651 ATCGACACTG CATTACGATG GAAAGATGAA AACCACGAAC CCGTGCAAGA TAGCTGTGAC GTAATGCTAC CTTTCTACTT TTGGTGCTTG GGCACGTTCT 2701 AGAACATTGA AATCGATATT ACAGGGGCCA CAAAGCCGAA GCCAGGGGAT TCTTGTAACT TTAGCTATAA TGTCCCCGGT GTTTCGGCTT CGGTCCCCTA 2751 ATCATCCTGA CATGTTTCCG CGGGTGGGTT AAGCAATTGC AAATCGACTA TAGTAGGACT GTACAAAGGC GCCCACCCAA TTCGTTAACG TTTAGCTGAT 2801 TCCCGGACAT GAAGTAATGA CAGCCGCGGC CTCACAAGGG CTAACCAGAA AGGGCCTGTA CTTCATTACT GTCGGCGCCG GAGTGTTCCC GATTGGTCTT 2851 AAGGAGTGTA TGCCGTCCGG CAAAAAGTCA ATGAAAACCC ACTGTACGCG TTCCTCACAT ACGGCAGGCC GTTTTTCAGT TACTTTTGGG TGACATGCGC 2901 ATCACATCAG AGCATGTGAA CGTGTTGCTC ACCCGCACTG AGGACAGGCT TAGTGTAGTC TCGTACACTT GCACAACGAG TGGGCGTGAC TCCTGTCCGA 2951 AGTGTGGAAA ACCTTGCAGG GCGACCCATG GATTAAGCAG CTCACTAACA TCACACCTTT TGGAACGTCC CGCTGGGTAC CTAATTCGTC GAGTGATTGT 3001 TACCTAAAGG AAACTTTCAG GCTACTATAG AGGACTGGGA AGCTGAACAC ATGGATTTCC TTTGAAAGTC CGATGATATC TCCTGACCCT TCGACTTGTG 3051 AAGGGAATAA TTGCTGCAAT AAACAGCCCC ACTCCCCGTG CCAATCCGTT TTCCCTTATT AACGACGTTA TTTGTCGGGG TGAGGGGCAC GGTTAGGCAA 3101 CAGCTGCAAG ACCAACGTTT GCTGGGCGAA AGCATTGGAA CCGATACTAG GTCGACGTTC TGGTTGCAAA CGACCCGCTT TCGTAACCTT GGCTATGATC 3151 CCACGGCCGG TATCGTACTT ACCGGTTGCC AGTGGAGCGA ACTGTTCCCA GGTGCCGGCC ATAGCATGAA TGGCCAACGG TCACCTCGCT TGACAAGGGT 3201 CAGTTTGCGG ATGACAAACC ACATTCGGCC ATTTACGCCT TAGACGTAAT GTCAAACGCC TACTGTTTGG TGTAAGCCGG TAAATGCGGA ATCTGCATTA 3251 TTGCATTAAG TTTTTCGGCA TGGACTTGAC AAGCGGACTG TTTTCTAAAC AACGTAATTC AAAAAGCCGT ACCTGAACTG TTCGCCTGAC AAAAGATTTG 3301 AGAGCATCCC ACTAACGTAC CATCCCGCCG ATTCAGCGAG GCCGGTAGCT TCTCGTAGGG TGATTGCATG GTAGGGCGGC TAAGTCGCTC CGGCCATCGA 3351 CATTGGGACA ACAGCCCAGG AACCCGCAAG TATGGGTACG ATCACGCCAT GTAACCCTGT TGTCGGGTCC TTGGGCGTTC ATACCCATGC TAGTGCGGTA 3401 TGCCGCCGAA CTCTCCCGTA GATTTCCGGT GTTCCAGCTA GCTGGGAAGG ACGGCGGCTT GAGAGGGCAT CTAAAGGCCA CAAGGTCGAT CGACCCTTCC 3451 GCACACAACT TGATTTGCAG ACGGGGAGAA CCAGAGTTAT CTCTGCACAG CGTGTGTTGA ACTAAACGTC TGCCCCTCTT GGTCTCAATA GAGACGTGTC 3501 CATAACCTGG TCCCGGTGAA CCGCAATCTT CCTCACGCCT TAGTCCCCGA GTATTGGACC AGGGCCACTT GGCGTTAGAA GGAGTGCGGA ATCAGGGGCT 3551 GTACAAGGAG AAGCAACCCG GCCCGGTCGA AAAATTCTTG AACCAGTTCA CATGTTCCTC TTCGTTGGGC CGGGCCAGCT TTTTAAGAAC TTGGTCAAGT 3601 AACACCACTC AGTACTTCTG GTATCAGAGG AAAAAATTGA ACCTCCCCCT TTGTGGTGAG TCATGAACAC CATAGTCTCC TTTTTTAACT TCGAGGGGCA 3651 AAGAGAATCG AATGGATCGC CCCGATTGGC ATAGCCGGTG CAGATAAGAA TTCTCTTAGC TTACCTAGCG GGGCTAACCG TATCGGCCAC GTCTATTCTT 3701 CTACAACCTG GCTTTCGGGT TTCCGCCGCA GGCACGGTAC GACCTGGTGT GATGTTGGAC CGAAAGCCCA AAGGCGGCGT CCGTGCCATG CTGGACCACA 3751 TCATCAACAT TGGAACTAAA TACAGAAACC ACCACTTTCA GCAGTGCGAA AGTAGTTGTA ACCTTGATTT ATGTCTTTGG TGGTGAAAGT CGTCACGCTT 3801 GACCATGCGG CGACCTTAAA AACCCTTTCG CGTTCGGCCC TGAATTGCCT CTGGTACGCC GCTGGAATTT TTGGGAAAGC GCAAGCCGGG ACTTAACGGA 3851 TAACCCAGGA GGCACCCTCG TGGTGAAGTC CTATGGCTAC GCCGACCGCA ATTGGGTCCT CCGTGGGAGC ACCACTTCAG GATACCGATG CGGCTGGCGT 3901 ACAGTGAGGA CGTAGTCACC GCTCTTGCCA GAAAGTTTGT CAGGGTGTCT TGTCACTCCT GCATCAGTGG CGAGAACGGT CTTTCAAACA GTCCCACAGA 3951 GCAGCGAGAC CAGATTGTGT CTCAAGCAAT ACAGAAATGT ACCTGATTTT CGTCGCTCTG GTCTAACACA GAGTTCGTTA TGTCTTTACA TGGACTAAAA 4001 CCGACAACTA GACAACAGCC GTACACGGCA ATTCACCCCG CACCATCTGA GGCTGTTGAT CTGTTGTCGG CATGTGCCGT TAAGTGGGGC GTGGTAGACT 4051 ATTGCGTGAT TTCGTCCGTG TATGAGGGTA CAAGAGATGG AGTTGGAGCC TAACGCACTA AAGCAGGCAC ATACTCCCAT GTTCTCTACC TCAACCTCGG 4101 GCGCCGTCAT ACCGCACCAA AAGGGAGAAT ATTGCTGACT GTCAAGAGGA CGCGGCAGTA TGGCGTGGTT TTCCCTCTTA TAACGACTGA CAGTTCTCCT 4151 AGCAGTTGTC AACGCAGCCA ATCCGCTGGG TAGACCAGGC GAAGGAGTCT TCGTCAACAG TTGCGTCGGT TAGGCGACCC ATCTGGTCCG CTTCCTCAGA 4201 GCCGTGCCAT CTATAAACGT TGGCCGACCA GTTTTACCGA TTCAGCCACG CGGCACGGTA GATATTTGCA ACCGGCTGGT CAAAATGGCT AAGTCGGTGC 4251 GAGACAGGCA CCGCAAGAAT GACTGTGTGC CTAGGAAAGA AAGTGATCCA CTCTGTCCGT GGCGTTCTTA CTGACACACG GATCCTTTCT TTCACTAGGT 4301 CGCGGTCGGC CCTGATTTCC GGAAGCACCC AGAAGCAGAA GCCTTGAAAT GCGCCAGCCG GGACTAAAGG CCTTCGTGGG TCTTCGTCTT CGGAACTTTA 4351 TGCTACAAAA CGCCTACCAT GCAGTGGCAG ACTTAGTAAA TGAACATAAC ACGATGTTTT GCGGATGGTA CGTCACCGTC TGAATCATTT ACTTGTATTG 4401 ATCA7GTCTG TCGCCATTCC ACTGCTATCT ACAGGCATTT ACGCAGCCGG TAGTTCAGAC AGCGGTAAGG TGACGATAGA TGTCCGTAAA TGCGTCGGCC 4451 AAAAGACCGC CTTGAAGTAT CACTTAACTG CTTGACAACC GCGCTAGACA TTTTCTGGCG CAACTTCATA GTGAATTGAC GAACTGTTGG CGCGATCTGT 4501 GAACTGACGC GGACGTAACC ATCTATTGCC TGGATAAGAA GTGGAAGGAA CTTGACTGCG CCTGCATTGG TAGATAACGG ACCTATTCTT CACCTTCCTT 4551 AGAATCGACG CGGCACTCCA ACTTAAGGAG TCTGTAACAG AGCTGAAGGA TCTTAGCTGC GCCGTGAGGT TGAATTCCTC AGACATTGTC TCGACTTCCT 4601 TGAAGATATG GAGATCGACG ATGAGTTAGT ATGGATCCAT CCAGACAGTT ACTTCTATAC CTCTAGCTGC TACTCAATCA TACCTAGGTA GGTCTGTCAA 4651 GCTTGAAGGG AAGAAAGGGA TTCAGTACTA CAAAAGGAAA ATTGTATTCG CGAACTTCCC TTCTTTCCCT AAGTCATGAT GTTTTCCTTT TAACATAAGC 4701 TACTTCGAAG GCACCAAATT CCATCAAGCA GCAAAAGACA TGGCGGAGAT ATGAAGCTTC CGTGGTTTAA GGTAGTTCGT CGTTTTCTGT ACCGCCTCTA 4751 AAAGGTCCTG TTCCCTAATG ACCAGGAAAG TAATGAACAA CTGTGTGCCT TTTCCAGGAC AAGGGATTAC TGGTCCTTTC ATTACTTGTT GACACACGGA 4801 ACATATTGGG TGAGACCATG GAAGCAATCC GCGAAAAGTG CCCGGTCGAC TGTATAACCC ACTCTGGTAC CTTCGTTAGG CGCTTTTCAC GGGCCAGCTG 4851 CATAACCCGT CGTCTAGCCC GCCCAAAACG TTGCCGTGCC TTTGCATGTA GTATTGGGCA GCAGATCGGG CGGGTTTTGC AACGGCACGG AAACGTACAT 4901 TGCCATGACG CCAGAAAGGG TCCACAGACT TAGAAGCAAT AACGTCAAAG ACGGTACTGC GGTCTTTCCC AGGTGTCTGA ATCTTCGTTA TTGCAGTTTC 4951 AAGTTACAGT ATGCTCCTCC ACCCCCCTTC CTAAGCACAA AATTAAGAAT TTCAATGTCA TACGAGGAGG TGGGGGGAAG GATTCGTGTT TTAATTCTTA 5001 GTTCAGAAGG TTCAGTGCAC GAAAGTAGTC CTGTTTAATC CGCACACTCC CAAGTCTTCC AAGTCACGTG CTTTCATCAG GACAAATTAG GCGTGTGAGG 5051 CGCATTCGTT CCCGCCCGTA AGTACATAGA AGTGCCAGAA CAGCCTACCG GCGTAAGCAA GGGCGGGCAT TCATGTATCT TCACGGTCTT GTCGGATGGC 5101 CTCCTCCTGC ACAGGCCGAG GAGGCCCCCG AAGTTGTAGC GACACCGTCA GAGGAGGACG TGTCCGGCTC CTCCGGCCGC TTCAACATCG CTGTGGCAGT 5151 CCATCTACAG CTGATAACAC CTCGCTTGAT GTCACAGACA TCTCACTGGA GGTAGATGTC GACTATTGTG GAGCCAACTA CAGTGTCTGT AGAGTGACCT 5201 TATGGATGAC AGTAGCGAAG GCTCACTTTT TTCGAGCTTT AGCGGATCGG ATACCTACTG TCATCGCTTC CGAGTGAAAA AAGCTCGAAA TCGCCTAGCC 5251 ACAACTCTAT TACTAGTGCC TCGTACCCCG GCCATCAACA CGCGTCTGCG TGTTGAGATA ATGATCACGG AGCATGGGGC CGGTAGTTGT GCGCAGACGC 5301 TTCGACCAGG CTGCGCGTTC TCGCGGCCAT AGCAACCGAC GTACGGCGTT AAGCTGGTCC GACGCGCAAG AGCGCCGGTA TCGTTGGCTG CATGCCGCAA 5351 GCGCCCTCGC CGGCAGCAAG AAGCCACGGA AGTCCGCCCG GAGCAGAAAA CGCGGGAGCG GCCGTCGTTC TTCGGTGCCT TCAGGCGGGC CTCGTCTTTT 5401 TGCCCACGCT ACTGCGGGTT TATATAGACG GTCCCCACGG GATGGGGAAA ACGGGTGCGA TGACGCCCAA ATATATCTGC CAGGGGTGCC CTACCCCTTT 5451 ACCACCACCA CGCAACTGCT GGTGGCCCTG GGTTCGCGCG ACGATATCGT TGGTGGTGGT GCGTTGACGA CCACCGGGAC CCAAGCGCGC TGCTATAGCA 5501 CTACGTACCC GAGCCGATGA CTTACTGGCG GGTGCTGGGG GCTTCCGAGA GATGCATGGG CTCGGCTACT GAATGACCGC CCACGACCCC CGAAGGCTCT 5551 CAATCGCGAA CATCTACACC ACACAACACC GCCTCGACCA GGGTGAGATA GTTAGCGCTT GTAGATGTGG TGTGTTGTGG CGGAGCTGGT CCCACTCTAT 5601 TCGGCCGGGG ACGCGGCGGT GGTAATGACA AGCGCCCAGA TAACAATGGG AGCCGGCCCC TGCGCCGCCA CCATTACTGT TCGCGGGTCT ATTGTTACCC 5651 CATGCCTTAT GCCGTGACCG ACGCCGTTCT GGCTCCTCAT ATCGGGGGGG GTACGGAATA CGGCACTGGC TGCGGCAAGA CCGAGGAGTA TAGCCCCCCC 5701 AGGCTGGGAG CTCACATGCC CCGCCCCCGG CCCTCACCCT CATCTTCGAC TCCGACCCTC GAGTGTACGG GGCGGGGGCC GGGAGTGGGA GTAGAAGCTG 5751 CGCCATCCCA TCGCCGCCCT CCTGTGCTAC CCGGCCGCGC GGTACCTTAT GCGGTAGGCT AGCGGCGGGA GGACACGATG GGCCGGCGCG CCATGGAATA 5801 GGGCAGCATG ACCCCCCAGG CCGTGCTGGC GTTCGTGGCC CTCATCCCGC CCCGTCGTAC TGGGGGGTCC GGCACGACCG CAAGCACCGG GAGTAGGGCG 5851 CGACCTTGCC CGGCACCAAC ATCGTGCTTG GGGCCCTTCC GGAGGACAGA GCTGGAACGG GCCGTGGTTG TAGCACGAAC CCCGGGAAGG CCTCCTGTCT 5901 CACATCGACC GCCTGGCCAA ACGCCAGCGC CCCGGCGAGC GGCTGGACCT GTGTAGCTGG CGGACCGGTT TGCGGTCGCG GGGCCGCTCG CCGACCTGGA 5951 GGCTATGCTG GCTGCGATTC GCCGCGTTTA CGGGCTACTT GCCAATACGG CCGATACGAC CGACGCTAAG CGGCGCAAAT GCCCGATGAA CGGTTATGCC 6001 TGCGGTATCT GCAGTGCGGC GGGTCGTGGC GGGAGGACTG GGGACAGCTT ACGCCATAGA CGTCACGCCG CCCAGCACCG CCCTCCTGAC CCCTGTCGAA 6051 TCGGGGACGG CCGTGCCGCC CCAGGGTGCC GAGCCCCAGA GCAACGCGGG AGCCCCTGCC GGCACGGCGG GGTCCCACGG CTCGGGGTCT CGTTGCGCCC 6101 CCCACGACCC CATATCGGGG ACACGTTATT TACCCTGTTT CGGGCCCCCG GGGTGCTGGG GTATAGCCCC TGTGCAATAA ATGGGACAAA GCCCGGGGGC 6151 AGTTGCTGGC CCCCAACGGC GACCTGTATA ACGTGTTTGC CTGGGCCTTG TCAACGACCG GGGGTTGCCG CTGGACATAT TGCACAAACG GACCCGGAAC 6201 GACGTCTTGG CCAAACGCCT CCGTTCCATG CACGTCTTTA TCCTGGATTA CTGCAGAACC GGTTTGCGGA GGCAAGGTAC GTGCAGAAAT AGGACCTAAT 6251 CGACCAATCG CCCGCCGGCT GCCGGGACGC CCTGCTGCAA CTTACCTCCG GCTGGTTAGC GGGCGGCCGA CGGCCCTGCG GGACGACGTT GAATGGAGGC 6301 GGATGGTCCA GACCCACGTC ACCACCCCCG GCTCCATACC GACGATATGC CCTACCAGGT CTGGGTGCAG TGGTGGGGGC CGAGGTATGG CTGCTATACG 6351 GACCTGGCGC GCACGTTTCC CCGGGAGATG GGGGAGGCTA ACACTAGTAT CTGGACCGCG CGTGCAAACG GGCCCTCTAC CCCCTCCGAT TGTGATCATA 6401 GGACAGTTGG TCGTCAGGAC CTAGTTCACT AGAGATAGTA GACCGAAGGC CCTGTCAACC AGCAGTCCTG GATCAAGTGA TCTCTATCAT CTGGCTTCCG 6451 AGGTGGTGGT GGCTGACGTT CATGCCGTCC AAGAGCCTGC CCCTATTCCA TCCACCACCA CCGACTGCAA GTACGGCAGG TTCTCGGACG GGGATAAGGT 6501 CCGCCAAGGC TAAAGAAGAT GGCCCGCCTG GCAGCGGCAA GAAAAGAGCC GGCGGTTCCG ATTTCTTCTA CCGGGCGGAC CGTCGCCGTT CTTTTCTCGG 6551 CACTCCACCG GCAAGCAATA GCTCTGAGTC CCTCCACCTC TCTTTTGGTG GTGAGGTGGC CGTTCGTTAT CGAGACTCAG GGAGGTGGAG AGAAAACCAC 6601 GGGTATCCAT GTCCCTCGGA TCAATTTTCG ACGGAGAGAC GGCCCGCCAG CCCATAGGTA CAGGGAGCCT AGTTAAAAGC TCCCTCTCTG CCGGGCGGTC 6651 GCAGCGGTAC AACCCCTGGC AACAGGCCCC ACGGATGTGC CTATGTCTTT CGTCGCCATG TTGGGGACCG TTGTCCGGGG TGCCTACACG GATACAGAAA 6701 CGGATCGTTT TCCGACGGAG AGATTGATGA GCTGAGCCGC AGAGTAACTG GCCTAGCAAA AGGCTGCCTC TCTAACTACT CGACTCGGCG TCTCATTGAC 6751 AGTCCGAACC CGTCCTGTTT GGATCATTTG AACCGGGCGA AGTGAACTCA TCAGGCTTGG GCAGGACAAA CCTAGTAAAC TTGGCCCGCT TCACTTGAGT 6801 ATTATATCGT CCCGATCAGC CGTATCTTTT CCACTACGCA AGCAGAGACG TAATATAGCA GGGCTAGTCG GCATAGAAAA GGTGATGCGT TCGTCTCTGC 6851 TAGACGCAGG AGCAGGAGGA CTGAATACTG ACTAACCGGG GTAGGTGGGT ATCTGCGTCC TCGTCCTCCT GACTTATGAC TGATTGGCCC CATCCACCCA 6901 ACATATTTTC GACGGACACA GGCCCTGGGC ACTTGCAAAA GAAGTCCGTT TGTATAAAAG CTGCCTGTGT CCGGGACCCG TGAACGTTTT CTTCAGGCAA 6951 CTGCAGAACC AGCTTACAGA ACCGACCTTG GAGCGCAATG TCCTGGAAAG GACGTCTTGG TCGAATGTCT TGGCTGGAAC CTCGCGTTAC AGGACCTTTC 7001 AATTCATGCC CCGGTGCTCG ACACGTCGAA AGAGGAACAA CTCAAACTCA TTAAGTACGG GGCCACGAGC TGTGCAGCTT TCTCCTTGTT GAGTTTGAGT 7051 GGTACCAGAT GATGCCCACC GAAGCCAACA AAAGTAGGTA CCAGTCTCGT CCATGGTCTA CTACGGGTGG CTTCGGTTGT TTTCATCCAT GGTCAGAGCA 7101 AAAGTAGAAA ATCAGAAAGC CATAACCACT GAGCGACTAC TGTCAGGACT TTTCATCTTT TAGTCTTTCG GTATTGGTGA CTCGCTGATG ACAGTCCTGA 7151 ACGACTGTAT AACTCTGCCA CAGATCAGCC AGAATGCTAT AAGATCACCT TGCTGACATA TTGAGACGGT GTCTAGTCGG TCTTACGATA TTCTAGTGGA 7201 ATCCGAAACC ATTGTACTCC AGTAGCGTAC CGGCGAACTA CTCCGATCCA TAGGCTTTGG TAACATGAGG TCATCGCATG GCCGCTTGAT GAGGCTAGGT 7251 CAGTTCGCTG TAGCTGTCTG TAACAACTAT CTGCATGAGA ACTATCCGAC GTCAAGCGAC ATCGACAGAC ATTGTTGATA GACGTACTCT TGATAGGCTG 7301 AGTAGCATCT TATCAGATTA CTGACGAGTA CGATGCTTAC TTGGATATGG TCATCGTAGA ATAGTCTAAT GACTGCTCAT GCTACGAATG AACCTATACC 7351 TAGACGGGAC AGTCGCCTGC CTGGATACTG CAACCTTCTG CCCCGCTAAG ATCTGCCCTG TCAGCGGACG GACCTATGAC GTTGGAAGAC GGGGCGATTC 7401 CTTAGAAGTT ACCCGAAAAA ACATGAGTAT AGAGCCCCGA ATATCCGCAG GAATCTTCAA TGGGCTTTTT TGTACTCATA TCTCGGGGCT TATAGGCGTC 7451 TGCGGTTCCA TCAGCGATGC AGAACACGCT ACAAAATGTG CTCATTGCCG ACGCCAAGGT AGTCGCTACG TCTTGTGCGA TGTTTTACAC GAGTAACGGC 7501 CAACTAAAAG AAATTGCAAC GTCACGCAGA TGCGTGAACT GCCAACACTG GTTGATTTTC TTTAACGTTG CAGTGCGTCT ACGCACTTGA CGGTTGTGAC 7551 GACTCAGCCA CATTCAATGT CGAATGCTTT CGAAAATATG CATGTAATGA CTGAGTCGCT GTAAGTTACA GCTTACGAAA GCTTTTATAC GTACATTACT 7601 CGAGTATTGG GAGGAGTTCG CTCGGAAGCC AATTAGGATT ACCACTGAGT GCTCATAACC CTCCTCAAGC GAGCCTTCGG TTAATCCTAA TGGTGACTCA 7651 TTGTCACCGC ATATGTAGCT AGACTGAAAG GCCCTAAGGC CGCCGCACTA AACAGTGGCG TATACATCGA TCTGACTTTC CGGGATTCCG GCGGCGTGAT 7701 TTTGCAAAGA CGTATAATTT GGTCCCATTG CAAGAAGTGC CTATGGATAG AAACGTTTCT GCATATTAAA CCAGGGTAAC GTTCTTCACG GATACCTATC 7751 ATTCGTCATG GACATGAAAA GAGACGTGAA AGTTACACCA GGCACGAAAC TAAGCAGTAC CTGTACTTTT CTCTGCACTT TCAATGTGGT CCGTGCTTTG 7801 ACACAGAAGA AAGACCGAAA GTACAAGTGA TACAAGCCGC AGAACCCCTG TGTGTCTTCT TTCTGGCTTT CATGTTCACT ATGTTCGGCG TCTTGGGGAC 7851 GCGACTGCTT ACTTATGCGG GATTCACCGG GAATTAGTGC GTAGGCTTAC CGCTGACGAA TGAATACGCC CTAAGTGGCC CTTAATCACG CATCCGAATG 7901 GGCCGTCTTG CTTCCAAACA TTCACACGCT TTTTGACATG TCGGCGGAGG CCGGCAGAAC GAAGGTTTGT AAGTGTGCGA AAAACTGTAC AGCCGCCTCC 7951 ATTTTGATGC AATCATAGCA GAACACTTCA AGCAAGGCGA CCCGGTACTG TAAAACTACG TTAGTATCGT CTTGTGAAGT TCGTTCCGCT GGGCCATGAC 8001 GAGACGGATA TCGCATCATT CGACAAAAGC CAAGACGACG CTATGGCGTT CTCTGCCTAT AGCGTAGTAA GCTGTTTTCG GTTCTGCTGC GATACCGCAA 8051 AACCGGTCTG ATGATCTTGG AGGACCTGGG TGTGGATCAA CCACTACTCG TTGGCCAGAC TACTAGAACC TCCTGGACCC ACACCTAGTT GGTGATGAGC 8101 ACTTGATCGA GTGCGCCTTT GGAGAAATAT CATCCACCCA TCTACCTACG TGAACTAGCT CACGCGGAAA CCTCTTTATA GTAGGTGGGT AGATGGATGC 8151 GGTACTCGTT TTAAATTCGG GGCGATGATG AAATCCGGAA TGTTCCTCAC CCATGAGCAA AATTTAAGCC CCGCTACTAC TTTAGGCCTT ACAAGGAGTG 8201 ACTTTTTGTC AACACAGTTT TGAATGTCGT TATCGCCAGC AGAGTACTAG TGAAAAACAG TTGTGTCAAA ACTTACAGCA ATAGCGGTCG TCTCATGATC 8251 AAGAGCGGCT TAAAACGTCC AGATGTGCAG CGTTCATTGG CGACGACAAC TTCTCGCCGA ATTTTGCAGG TCTACACGTC GCAAGTAACC GCTGCTGTTG 8301 ATCATACATG GAGTAGTATC TGACAAAGAA ATGGCTGAGA GGTGCGCCAC TAGTATGTAC CTCATCATAG ACTGTTTCTT TACCGACTCT CCACGCGGTG 8351 CTGGCTCAAC ATGGAGGTTA AGATCATCGA CGCAGTCATC GGTGAGAGAC GACCGAGTTG TACCTCCAAT TCTAGTAGCT GCGTCAGTAG CCACTCTCTG 8401 CACCTTACTT CTGCGGCGGA TTTATCTTGC AAGATTCGGT TACTTCCACA GTGGAATGAA GACGCCGCCT AAATAGAACG TTCTAAGCCA ATGAAGGTGT 8451 GCGTGCCGCG TGGCGGACCC CCTGAAAAGG CTGTTTAAGT TGGGTAAACC CGCACGGCGC ACCGCCTGGG GGACTTTTCC GACAAATTCA ACCCATTTGG 8501 GCTCCCAGCC GACGACGAGC AAGACGAAGA CAGAAGACGC GCTCTGCTAG CGAGGGTCGG CTGCTGCTCG TTCTGCTTCT GTCTTCTGCG CGAGACGATC 8551 ATGAAACAAA GGCGTGGTTT AGAGTAGGTA TAACAGGCAC TTTAGCAGTG TACTTTGTTT CCGCACCAAA TCTCATCCAT ATTGTCCGTG AAATCGTCAC 8601 GCCGTGACGA CCCGGTATGA GGTAGACAAT ATTACACCTG TCCTACTGGC CGGCACTGCT GGGCCATACT CCATCTGTTA TAATGTGGAC AGGATGACCG 8651 ATTGAGAACT TTTGCCCAGA GCAAAAGAGC ATTCCAAGCC ATCAGAGGGG TAACTCTTGA AAACGGGTCT CGTTTTCTCG TAAGGTTCGG TAGTCTCCCC 8701 AAATAAAGCA TCTCTACGGT GGTCCTAAAT AGTCAGCATA GTACATTTCA TTTATTTCGT AGAGATGCCA CCAGGATTTA TCAGTCGTAT GATGTAAAGT 8751 TCTGACTAAT ACTACAACAC CACCACCTCT AGCCCGGGCT CGAGATCTGC AGACTGATTA TGATGTTGTG GTGGTGGAGA TCGGGCCCGA GCTCTAGACG 8801 GATCTAAGTA AGCTTGGCAT TCCGGTACTG TTGGTAAAGC CACCATGGAA CTAGATTCAT TCGAACCGTA AGGCCATGAC AACCATTTCG GTGGTACCTT 8851 GACGCCAAAA ACATAAAGAA AGGCCCGGCG CCATTCTATC CGCTGGAAGA CTGCGGTTTT TGTATTTCTT TCCGGGCCGC GGTAAGATAG GCGACCTTCT 8901 TGGAACCGCT GGAGAGCAAC TGCATAAGGC TATGAAGAGA TACGCCCTGG ACCTTGGCGA CCTCTCGTTG ACGTATTCCG ATACTTCTCT ATGCGGGACC 8951 TTCCTGGAAC AATTGCTTTT ACAGATGCAC ATATCGAGGT GGACATCACT AAGGACCTTG TTAACGAAAA TGTCTACGTG TATAGCTCCA CCTGTAGTGA 9001 TACGCTGAGT ACTTCGAAAT GTCCGTTCGG TTGGCAGAAG CTATGAAACG ATGCGACTCA TGAAGCTTTA CAGGCAAGCC AACCGTCTTC GATACTTTGC 9051 ATATGGGCTG AATACAAATC ACAGAATCGT CGTATGCAGT GAAAACTCTC TATACCCGAC TTATGTTTAG TGTCTTAGCA GCATACGTCA CTTTTGAGAG 9101 TTCAATTCTT TATGCCGGTG TTGGGCGCGT TATTTATCGG AGTTGCAGTT AAGTTAAGAA ATACGGCCAC AACCCGCGCA ATAAATAGCC TCAACGTCAA 9151 GCGCCCGCGA ACGACATTTA TAATGAACGT GAATTGCTCA ACAGTATGGG CGCGGGCGCT TGCTGTAAAT ATTACTTGCA CTTAACGAGT TGTCATACCC 9201 CATTTCGCAG CCTACCGTGG TGTTCGTTTC CAAAAAGGGG TTGCAAAAAA GTAAAGCGTC GGATGGCACC ACAAGCAAAG GTTTTTCCCC AACGTTTTTT 9251 TTTTGAACGT GCAAAAAAAG CTCCCAATCA TCCAAAAAAT TATTATCATG AAAACTTGCA CGTTTTTTTC GAGGGTTAGT AGGTTTTTTA ATAATAGTAC 9301 GATTCTAAAA CGGATTACCA GGGATTTCAG TCGATGTACA CGTTCGTCAC CTAAGATTTT GCCTAATGGT CCCTAAAGTC AGCTACATGT GCAAGCAGTG 9351 ATCTCATCTA CCTCCCGGTT TTAATGAATA CGATTTTGTG CCAGAGTCCT TAGAGTAGAT GGAGGGCCAA AATTACTTAT GCTAAAACAC GGTCTCAGGA 9401 TCGATAGGGA CAAGACAATT GCACTGATCA TGAACTCCTC TGGATCTACT AGCTATCCCT GTTCTGTTAA CGTGACTAGT ACTTGAGGAG ACCTAGATGA 9451 GGTCTGCCTA AAGGTGTCGC TCTGCCTCAT AGAACTGCCT GCGTGAGATT CCAGACGGAT TTCCACAGCG AGACGGAGTA TCTTGACGGA CGCACTCTAA 9501 CTCGCATGCC AGAGATCCTA TTTTTGGCAA TCAAATCATT CCGGATACTG GAGCGTACGG TCTCTAGGAT AAAAACCGTT AGTTTAGTAA GGCCTATGAC 9551 CGATTTTAAG TGTTGTTCCA TTCCATCACG GTTTTGGAAT GTTTACTACA GCTAAAATTC ACAACAAGGT AAGGTAGTGC CAAAACCTTA CAAATGATGT 9601 CTCGGATATT TGATATGTGG ATTTCGAGTC GTCTTAATGT ATAGATTTGA GAGCCTATAA ACTATACACC TAAAGCTCAG CAGAATTACA TATCTAAACT 9651 AGAAGAGCTG TTTCTGAGGA GCCTTCAGGA TTACAAGATT CAAAGTGCGC TCTTCTCGAC AAAGACTCCT CGGAAGTCCT AATGTTCTAA GTTTCACGCG 9701 TGCTGGTGCC AACCCTATTC TCCTTCTTCG CCAAAAGCAC TCTGATTGAC ACGACCACGG TTGGGATATG AGGAAGAAGC GGTTTTCGTG AGACTAACTG 9751 AAATACGATT TATCTAATTT ACACGAAATT GCTTCTGGTG GCGCTCCCCT TTTATGCTAA ATAGATTAAA TGTGCTTTAA CGAAGACCAC CGCGAGGGGA 9801 CTCTAAGGAA GTCGGGGAAG CGGTTGCCAA GAGGTTCCAT CTGCCAGGTA GAGATTCCTT CAGCCCCTTC GCCAACGGTT CTCCAAGGTA GACGGTCCAT 9851 TCAGGCAAGG ATATGGGCTC ACTGAGACTA CATCAGCTAT TCTGATTACA AGTCCGTTCC TATACCCGAG TGACTCTGAT GTAGTCGATA AGACTAATGT 9901 CCCGAGGGGG ATGATAAACC GGGCGCGGTC GGTAAAGTTG TTCCATTTTT GGGCTCCCCC TACTATTTGG CCCGCGCCAG CCATTTCAAC AAGGTAAAAA 9951 TGAAGCGAAG GTTGTGGATC TGGATACCGG GAAAACGCTG GGCGTTAATC ACTTCGCTTC CAACACCTAG ACCTATGGCC CTTTTGCGAC CCGCAATTAG 10001 AAAGAGGCGA ACTGTGTGTG AGAGGTCCTA TGATTATGTC CGGTTATGTA TTTCTCCGCT TGACACACAC TCTCCAGGAT ACTAATACAG GCCAATACAT 10051 AACAATCCGG AAGCGACCAA CGCCTTGATT GACAAGGATG GATGGCTACA TTGTTAGGCC TTCGCTGGTT GCGGAACTAA CTGTTCCTAC CTACCGATGT 10101 TTCTGGAGAC ATAGCTTACT GGGACGAAGA CGAACACTTC TTCATCGTTG AAGACCTCTG TATCGAATGA CCCTGCTTCT GCTTGTGAAG AAGTAGCAAC 10151 ACCGCCTGAA GTCTCTGATT AAGTACAAAG GCTATCAGGT GGCTCCCGCT TGGCGGACTT CAGAGACTAA TTCATGTTTC CGATAGTCCA CCGAGGGCGA 10201 GAATTGGAAT CCATCTTGCT CCAACACCCC AACATCTTCG ACGCAGGTGT CTTAACCTTA GGTAGAACGA GGTTGTGGGG TTGTAGAAGC TGCGTCCACA 10251 CGCAGGTCTT CCCGACGATG ACGCCGGTGA ACTTCCCGCC GCCGTTGTTG GCGTCCAGAA GGGCTGCTAC TGCGGCCACT TGAAGGGCGG CGGCAACAAC 10301 TTTTGGAGCA CGGAAAGACG ATGACGGAAA AAGAGATCGT GGATTACGTC AAAACCTCGT GCCTTTCTGC TACTGCCTTT TTCTCTAGCA CCTAATGCAG 10351 GCCAGTCAAG TAACAACCGC GAAAAAGTTG CGCGGAGGAG TTGTGTTTGT CGGTCAGTTC ATTGTTGGCG CTTTTTCAAC GCGCCTCCTC AACACAAACA 10401 GGACGAAGTA CCGAAAGGTC TTACCGGAAA ACTCGACGCA AGAAAAATCA CCTGCTTCAT GGCTTTCCAG AATGGCCTTT TGAGCTGCGT TCTTTTTAGT 10451 GAGAGATCCT CATAAAGGCC AAGAAGGGCG GAAAGATCGC CGTGTAATTC CTCTCTAGGA GTATTTCCGG TTCTTCCCGC CTTTCTAGCG GCACATTAAG 10501 TAGAGGCGCG CCGATCTCAC GATCCCCTGA AAAGGCTGTT TAAGTTGGGT ATCTCCGCGC GGCTAGAGTG CTAGGGGACT TTTCCGACAA ATTCAACCCA 10551 AAACCGCTCC CAGCCGACGA CGAGCAAGAC GAAGACAGAA GACGCGCTCT TTTGGCGAGG GTCGGCTGCT GCTCGTTCTG CTTCTGTCTT CTGCGCGAGA 10601 GCTAGATGAA ACAAAGGCGT GGTTTAGAGT AGGTATAACA GGCACTTTAG CGATCTACTT TGTTTCCGCA CCAAATCTCA TCCATATTGT CCGTGAAATC 10651 CAGTGGCCGT GACGACCCGG TATGAGGTAG ACAATATTAC ACCTGTCCTA GTCACCGGCA CTGCTGGGCC ATACTCCATC TGTTATAATG TGGACAGGAT 10701 CTGGCATTGA GAACTTTTGC CCAGAGCAAA AGAGCATTCC AAGCCATCAG GACCGTAACT CTTGAAAACG GGTCTCGTTT TCTCGTAAGG TTCGGTAGTC 10751 AGGGGAAATA AAGCATCTCT ACGGTGGTCC TAAATAGTCA GCATAGTACA TCCCCTTTAT TTCGTAGAGA TGCCACCAGG ATTTATCAGT CGTATCATGT 10801 TTTCATCTGA CTAATACTAC AACACCACCA CCATGAATAG AGGATTCTTT AAAGTAGACT GATTATGATG TTGTGGTGGT GGTACTTATC TCCTAAGAAA 10851 AACATGCTCG GCCGCCGCCC CTTCCCGGCC CCCACTGCCA TGTGGAGGCC TTGTACGAGC CGGCGGCGGG GAAGGGCCGG GGGTGACGGT ACACCTCCGG 10901 GCGGAGAAGG AGGCAGGCGG CCCCGATGCC TGCCCGCAAC GGGCTGGCTT CGCCTCTTCC TCCGTCCGCC GGGGCTACGG ACGGGCGTTG CCCGACCGAA 10951 CTCAAATCCA GCAACTGACC ACAGCCGTCA GTGCCCTAGT CATTGGACAG GAGTTTAGGT CGTTGACTGG TGTCGGCAGT CACGGGATCA GTAACCTGTC 11001 GCAACTAGAC CTCAACCCCC ACGTCCACGC CAGCCACCGC GCCAGAAGAA CGTTGATCTG GAGTTGGGGG TGCAGGTGCG GTCGGTGGCG CGGTCTTCTT 11051 GCAGGCGCCC AAGCAACCAC CGAAGCCGAA GAAACCAAAA ACGCAGGAGA CGTCCGCGGG TTCGTTGGTG GCTTCGGCTT CTTTGGTTTT TGCGTCCTCT 11101 AGAAGAAGAA GCAACCTGCA AAACCCAAAC CCGGAAAGAG ACAGCGCATG TCTTCTTCTT CGTTGGACGT TTTGGGTTTG GGCCTTTCTC TGTCGCGTAC 11151 GCACTTAAGT TGGAGGCCGA CAGATTGTTC GACGTCAAGA ACGAGGACGG CGTGAATTCA ACCTCCGGCT GTCTAACAAG CTGCAGTTCT TGCTCCTGCC 11201 AGATGTCATC GGGCACGCAC TGGCCATGGA AGGAAAGGTA ATGAAACCTC TCTACAGTAG CCCGTGCGTG ACCGGTACCT TCCTTTCCAT TACTTTGGAG 11251 TGCACGTGAA AGGAACCATC GACCACCCTG TGCTATCAAA GCTCAAATTT ACGTGCACTT TCCTTGGTAG CTGGTGGGAC ACGATAGTTT CGAGTTTAAA 11301 ACCAAGTCGT CAGCATACGA CATGGAGTTC GCACAGTTGC CAGTCAACAT TGGTTCAGCA GTCGTATGCT GTACCTCAAG CGTGTCAACG GTCAGTTGTA 11351 GAGAAGTGAG GCATTCACCT ACACCAGTGA ACACCCCGAA GGATTCTATA CTCTTCACTC CGTAAGTGGA TGTGGTCACT TGTGGGGCTT CCTAAGATAT 11401 ACTGGCACCA CGGAGCGGTG CAGTATAGTG GAGGTAGATT TACCATCCCT TGACCGTGGT GCCTCGCCAC GTCATATCAC CTCCATCTAA ATGGTAGGGA 11451 CGCGGAGTAG GAGGCAGAGG AGACAGCGGT CGTCCGATCA TGGATAACTC GCGCCTCATC CTCCGTCTCC TCTGTCGCCA GCAGGCTAGT ACCTATTGAG 11501 CGGTCGGGTT GTCGCGATAG TCCTCGGTGG AGCTGATGAA GGAACACGAA GCCAGCCCAA CAGCGCTATC AGGAGCCACC TCGACTACTT CCTTGTGCTT 11551 CTGCCCTTTC GGTCGTCACC TGGAATAGTA AAGGGAAGAC AATTAAGACG GACGGGAAAG CCAGCAGTGG ACCTTATCAT TTCCCTTCTG TTAATTCTGC 11601 ACCCCGGAAG GGACAGAAGA GTGGTCCGCA GCACCACTGG TCACGGCAAT TGGGGCCTTC CCTGTCTTCT CACCAGGCGT CGTGGTGACC AGTGCCGTTA 11651 GTGTTTGCTC GGAAATGTGA GCTTCCCATG CGACCGCCCG CCCACATGCT CACAAACGAG CCTTTACACT CGAAGGGTAC GCTGGCGGGC GGGTGTACGA 11701 ATACCCGCGA ACCTTCCAGA GCCCTCGACA TCCTTGAAGA GAACGTGAAC TATGGGCGCT TGGAAGGTCT CGGGAGCTGT AGGAACTTCT CTTGCACTTG 11751 CATGAGGCCT ACGATACCCT GCTCAATGCC ATATTGCGGT GCGGATCGTC GTACTCCGGA TGCTATGGGA CGAGTTACGG TATAACGCCA CGCCTAGCAG 11801 TGGCAGAAGC AAAAGAAGCG TCATCGATGA CTTTACCCTG ACCAGCCCCT ACCGTCTTCG TTTTCTTCGC AGTAGCTACT GAAATGGGAC TGGTCGGGGA 11851 ACTTGGGCAC ATGCTCGTAC TGCCACCATA CTGAACCGTG CTTCAGCCCT TGAACCCGTG TACGAGCATG ACGGTGGTAT GACTTGGCAC GAAGTCGGGA 11901 GTTAAGATCG AGCAGGTCTG GGACGAAGCG GACGATAACA CCATACGCAT CAATTCTAGC TCGTCCAGAC CCTGCTTCGC CTGCTATTGT GGTATGCGTA 11951 ACAGACTTCC GCCCAGTTTG GATACGACCA AAGCGGAGCA GCAAGCGCAA TGTCTGAAGG CGGGTCAAAC CTATGCTGGT TTCGCCTCGT CGTTCGCGTT 12001 ACAAGTACCG CTACATGTCG CTTAAGCAGG ATCACACCGT TAAAGAAGGC TGTTCATGGC GATGTACAGC GAATTCGTCC TAGTGTGGCA ATTTCTTCCG 12051 ACCATGGATG ACATCAAGAT TAGCACCTCA GGACCGTGTA GAAGGCTTAG TGGTACCTAC TGTAGTTCTA ATCGTGGAGT CCTGGCACAT CTTCCGAATC 12101 CTACAAAGGA TACTTTCTCC TCGCAAAATG CCCTCCAGGG GACAGCGTAA GATGTTTCCT ATGAAAGAGG AGCGTTTTAC GGGAGGTCCC CTGTCGCATT 12151 CGGTTAGCAT AGTGAGTAGC AACTCAGCAA CGTCATGTAC ACTGGCCCGC GCCAATCGTA TCACTCATCG TTGAGTCGTT GCAGTACATG TGACCGGGCG 12201 AAGATAAAAC CAAAATTCGT GGGACGGGAA AAATATGATC TACCTCCCGT TTCTATTTTG GTTTTAAGCA CCCTGCCCTT TTTATACTAG ATGGAGGGCA 12251 TCACGGTAAA AAAATTCCTT GCACAGTGTA CGACCGTCTG AAAGAAACAA AGTGCCATTT TTTTAAGGAA CGTGTCACAT GCTGGCAGAC TTTCTTTGTT 12301 CTGCAGGCTA CATCACTATG CACAGGCCGG GCCCGCACGC TTATACATCC GACGTCCGAT GTAGTGATAC GTGTCCGGCC CGGGCGTGCG AATATGTAGG 12351 TACCTGGAAG AATCATCAGG GAAAGTTTAC GCAAAGCCGC CATCTGGGAA ATGGACCTTC TTAGTAGTCC CTTTCAAATG CGTTTCGGCG GTAGACCCTT 12401 GAACATTACG TATGAGTGCA AGTGCGGCGA CTACAAGACC GGAACCGTTT CTTGTAATGC ATACTCACGT TCACGCCGCT GATGTTCTGG CCTTGGCAAA 12451 CGACCCGCAC CGAAATCACT GGTTGCACCG CCATCAAGCA GTGCGTCGCC GCTGGGCGTG GCTTTAGTGA CCAACGTGGC GGTAGTTCGT CACGCAGCGG 12501 TATAAGAGCG ACCAAACGAA GTGGGTCTTC AACTCACCGG ACTTGATCCG ATATTCTCGC TGGTTTGCTT CACCCAGAAG TTGAGTGGCC TGAACTAGGC 12551 ACATGACGAC CACACGGTCC AAGGGAAATT GCATTTGCCT TTCAAGTTGA TGTACTGCTG GTGTGCCAGG TTCCCTTTAA CGTAAACGGA AAGTTCAACT 12601 TCCCGAGTAC CTGCATGGTC CCTGTTGCCC ACGCGCCGAA TGTAATACAT AGGGCTCATG GACGTACCAG GGACAACGGG TGCGCGGCTT ACATTATGTA 12651 GGCTTTAAAC ACATCAGCCT CCAATTAGAT ACAGACCACT TGACATTGCT CCGAAATTTG TGTAGTCGGA GGTTAATCTA TGTCTGGTGA ACTGTAACGA 12701 CACCACCAGG AGACTAGGGG CAAACCCGGA ACCAACCACT GAATGGATCG GTGGTGGTCC TCTGATCCCC GTTTGGGCCT TGGTTGGTGA CTTACCTAGC 12751 TCGGAAAGAC GGTCAGAAAC TTCACCGTCG ACCGAGATGG CCTGGAATAC AGCCTTTCTG CCAGTCTTTG AAGTGGCAGC TGGCTCTACC GGACCTTATG 12801 ATATGGGGAA ATCATGAGCC AGTGAGGGTC TATGCCCAAG AGTCAGCACC TATACCCCTT TAGTACTCGG TCACTCCCAG ATACGGGTTC TCAGTCGTGG 12851 AGGAGACCCT CACGGATGGC CACACGAAAT AGTACAGCAT TACTACCATC TCCTCTGGGA GTGCCTACCG GTGTGCTTTA TCATGTCGTA ATGATGGTAG 12901 GCCATCCTGT GTACACCATC TTAGCCGTCG CATCAGCTAC CGTGGCGATG CGGTAGGACA CATGTGGTAG AATCGGCAGC GTAGTCGATG GCACCGCTAC 12951 ATGATTGGCG TAACTGTTGC AGTGTTATGT GCCTGTAAAG CGCGCCGTGA TACTAACCGC ATTGACAACG TCACAATACA CGGACATTTC GCGCGGCACT 13001 GTGCCTGACG CCATACGCCC TGGCCCCAAA CGCCGTAATC CCAACTTCGC CACGGACTGC GGTATGCGGG ACCGGGGTTT GCGGCATTAG GGTTGAAGCG 13051 TGGCACTCTT GTGCTGCGTT AGGTCGGCCA ATGCTGAAAC GTTCACCGAG ACCGTGAGAA CACGACGCAA TCCAGCCGGT TACGACTTTG CAAGTGGCTC 13101 ACCATGAGTT ACTTGTGGTC GAACAGTCAG CCGTTCTTCT GGGTCCAGTT TGGTACTCAA TGAACACCAG CTTGTCAGTC GGCAAGAAGA CCCAGGTCAA 13151 GTGCATACCT TTGGCCGCTT TCATCGTTCT AATGCGCTGC TGCTCCTGCT CACGTATGGA AACCGGCGAA AGTAGCAAGA TTACGCGACG ACGAGGACGA 13201 GCCTGCCTTT TTTAGTGGTT GCCGGCGCCT ACCTGGCGAA GGTAGACGCC CGGACGGAAA AAATCACCAA CGGCCGCGGA TGGACCGCTT CCATCTGCGG 13251 TACGAACATG CGACCACTGT TCCAAATGTG CCACAGATAC CGTATAAGGC ATGCTTGTAC GCTGGTGACA AGGTTTACAC GGTGTCTATG GCATATTCCG 13301 ACTTGTTGAA AGGGCAGGGT ATGCCCCGCT CAATTTGGAG ATCACTGTCA TGAACAACTT TCCCGTCCCA TACGGGGCGA GTTAAACCTC TAGTGACAGT 13351 TGTCCTCGGA GGTTTTGCCT TCCACCAACC AAGAGTACAT TACCTGCAAA ACAGGAGCCT CCAAAACGGA AGGTGGTTGG TTCTCATGTA ATGGACGTTT 13401 TTCACCACTG TGGTCCCCTC CCCAAAAATC AAATGCTGCG GCTCCTTGGA AAGTGGTGAC ACCAGGGGAG GGGTTTTTAG TTTACGACGC CGAGGAACCT 13451 ATGTCAGCCG GCCGTTCATG CAGACTATAC CTGCAAGGTC TTCGGAGGGG TACAGTCGGC CGGCAAGTAC GTCTGATATG GACGTTCCAG AAGCCTCCCC 13501 TCTACCCCTT TATGTGGGGA GGAGCGCAAT GTTTTTGCGA CAGTGAGAAC AGATGOGGAA ATACACCCCT CCTCGCGTTA CAAAAACGCT GTCACTCTTG 13551 AGCCAGATGA GTGAGGCGTA CGTCGAACTG TCAGCAGATT GCGCGTCTGA TCGGTCTACT CACTCCGCAT GCAGCTTGAC AGTCGTCTAA CGCGCAGACT 13601 CCACGCGCAG GCGATTAAGG TGCACACTGC CGCGATGAAA GTAGGACTGC GGTGCGCGTC CGCTAATTCC ACGTGTGACG GCGCTACTTT CATCCTGACG 13651 GTATAGTGTA CGGGAACACT ACCAGTTTCC TAGATGTGTA CGTGAACGGA CATATCACAT GCCCTTGTGA TGGTCAAAGG ATCTACACAT GCACTTGCCT 13701 GTCACACCAG GAACGTCTAA AGACTTGAAA GTCATAGCTG GACCAATTTC CAGTGTGGTC CTTGCAGATT TCTGAACTTT CAGTATCGAC CTGGTTAAAG 13751 AGCATCGTTT ACGCCATTCG ATCATAAGGT CGTTATCCAT CGCGGCCTCG TCGTAGCAAA TGCGGTAAGC TAGTATTCCA GCAATAGGTA GCGCCGGACC 13801 TGTACAACTA TGACTTCCCG GAATATGGAG CGATGAAACC AGGAGCGTTT ACATGTTGAT ACTGAAGGGC CTTATACCTC GCTACTTTGG TCCTCGCAAA 13851 GGAGACATTC AAGCTACCTC CTTGACTAGC AAGGATCTCA TCGCCAGCAC CCTCTGTAAG TTCGATGGAG GAACTGATCG TTCCTAGAGT AGCGGTCGTG 13901 AGACATTAGG CTACTCAAGC CTTCCGCCAA GAACGTGCAT GTCCCGTACA TCTGTAATCC GATGAGTTCG GAAGGCGGTT CTTOCACOTA CAGGGCATGT 13951 CGCAGGCCGC ATCAGGATTT GAGATGTGGA AAAACAACTC AGGCCGCCCA GCGTCCGGCG TAGTCCTAAA CTCTACACCT TTTTGTTGAG TCCGGCGGGT 14001 CTGCAGGAAA CCGCACCTTT CGGGTGTAAG ATTGCAGTAA ATCCGCTCCG GACGTCCTTT GGCGTGGAAA GCCCACATTC TAACGTCATT TAGGCGAGGC 14051 AGCGGTGGAC TGTTCATACG GGAACATTCC CATTTCTATT GACATCCCGA TCGCCACCTG ACAAGTATGC CCTTGTAAGG GTAAAGATAA CTGTAGGGCT 14101 ACGCTGCCTT TATCAGGACA TCAGATGCAC CACTGGTCTC AACAGTCAAA TGCGACGGAA ATAGTCCTGT AGTCTACGTG GTGACCAGAG TTGTCAGTTT 14151 TGTGAAGTCA GTGAGTGCAC TTATTCAGCA GACTTCGGCG GGATGGCCAC ACACTTCAGT CACTCACGTG AATAAGTCGT CTGAAGCCGC CCTACCGGTG 14201 CCTGCAGTAT GTATCCGACC GCGAAGGTCA ATGCCCCGTA CATTCGCATT GGACGTCATA CATAGGCTGG CGCTTCCAGT TACGGGGCAT GTAAGCGTAA 14251 CGAGCACAGC AACTCTCCAA GAGTCGACAG TACATGTCCT GGAGAAAGGA GCTCGTGTCG TTGAGAGGTT CTCAGCTGTC ATGTACAGGA CCTCTTTCCT 14301 GCGGTGACAG TACACTTTAG CACCGCGAGT CCACAGGCGA ACTTTATCGT CGCCACTGTC ATGTGAAATC GTGGCGCTCA GGTGTCCGCT TGAAATAGCA 14351 ATCGCTGTGT GGGAAGAAGA CAACATGCAA TGCAGAATGT AAACCACCAG TAGCGACACA CCCTTCTTCT GTTGTACGTT ACGTCTTACA TTTGGTGGTC 14401 CTGACCATAT CGTGAGCACC CCGCACAAAA ATGACCAAGA ATTTCAAGCC GACTGGTATA GCACTCGTGG GGCGTGTTTT TACTGGTTCT TAAAGTTCGG 14451 GCCATCTCAA AAACATCATG GAGTTGGCTG TTTGCCCTTT TCGGCGGCGC CGGTAGAGTT TTTGTAGTAC CTCAACCGAC AAACGGGAAA AGCCGCCGCG 14501 CTCGTCGCTA TTAATTATAG GACTTATGAT TTTTGCTTGC AGCATGATGC GAGCAGCGAT AATTAATATC CTGAATACTA AAAACGAACG TCGTACTACG 14551 TGACTAGCAC ACGAAGATGA CCGCTACGCC CCAATGATCC GACCAGCAAA ACTGATCGTG TGCTTCTACT GGCGATGCGG GGTTACTAGG CTGGTCGTTT 14601 ACTCGATGTA CTTCCGAGGA ACTGATGTGC ATAAGTGAGC ATGCGTTTAA TGAGCTACAT GAAGGCTCCT TGACTACACG TATTCACTCG TACGCAAATT 14651 ACTGGGCCCA ATGTTCCCCA ATGATCCGAC CAGCAAAACT CGATGTACTT TGACCCGGGT TACAAGGGGT TACTAGGCTG GTCGTTTTGA GCTACATGAA 14701 CCGAGGAACT GATGTGCATA ATGCATCAGG CTGGTACATT AGATCCCCGC GGCTCCTTGA CTACACGTAT TACGTAGTCC GACCATGTAA TCTAGGGGCG 14751 TTACCGCGGG CAATATAGCA ACACTAAAAA CTCGATGTAC TTCCGAGGAA AATGGCGCCC GTTATATCGT TGTGATTTTT GAGCTACATG AAGGCTCCTT 14801 GCGCAGTGCA TAATGCTGCG CAGTGTTGCC ACATAACCAC TATATTAACC CGCGTCACGT ATTACGACGC GTCACAACGG TGTATTGGTG ATATAATTGG 14851 ATTTATCTAG CGGACGCCAA AA2CTCAATG TATTTCTGAG GAAGCGTGGT TAAATAGATC GCCTGCGGTT TTTGAGTTAC ATAAAGACTC CTTCGCACCA 14901 GCATAATGCC ACGCAGCGTC TGCATAACTT TTATTATTTC TTTTATTAAT CGTATTACGG TGCGTCGCAG ACGTATTGAA AATAATAAAG AAAATAATTA 14951 CAACAAAATT TTGTTTTTAA CATTTCAAAA AAAAAAAAAA AAAAAAAAAA GTTGTTTTAA AACAAAAATT GTAAAGTTTT TTTTTTTTTT TTTTTTTTTT 15001 AAAAAAAAAA AAAATTTAAA TTAATTAAGC GGCCGCCTCG AGGACGTCAG TTTTTTTTTT TTTTAAATTT AATTAATTCG CCGGCGGAGC TCCTGCAGTC 15051 GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC CACCGTGAAA AGCCCCTTTA CACGCGCCTT GGGGATAAAC AAATAAAAAG 15101 TAAATACATT CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT ATTTATGTAA GTTTATACAT AGGCGAGTAC TCTGTTATTG GGACTATTTA 15151 GCTTCAATAA TATTGAAAAA GGAAGAGTAT GAGTATTCAA CATTTCCGTG CGAAGTTATT ATAACTTTTT CCTTCTCATA CTCATAAGTT GTAAAGGCAC 15201 TCGCCCTTAT TCCCTTTTTT GCGGCATTTT GCCTTCCTGT TTTTGCTCAC AGCGGGAATA AGGGAAAAAA CGCCGTAAAA CGGAAGGACA AAAACGAGTG 15251 CCAGAAACGC TGGTGAAAGT AAAAGATGCT GAAGATCAGT TGGGTGCACG GGTCTTTGCG ACCACTTTCA TTTTCTACGA CTTCTAGTCA ACCCACGTGC 15301 AGTGGGTTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC CTTGAGAGTT TCACCCAATG TAGCTTGACC TAGAGTTGTC GCCATTCTAG GAACTCTCAA 15351 TTCGCCCCGA AGAACGTTTT CCAATGATGA GCACTTTTAA AGTTCTGCTA AAGCGGGGCT TCTTGCAAAA GGTTACTACT CGTGAAAATT TCAAGACGAT 15401 TGTGGCGCGG TATTATCCCG TATTGACGCC GGGCAAGAGC AACTCGGTCG ACACCGCGCC ATAATAGGGC ATAACTGCGG CCCGTTCTCG TTGAGCCAGC 15451 CCGCATACAC TATTCTCAGA ATGACTTGGT TGAGTACTCA CCAGTCACAG GGCGTATGTG ATAAGAGTCT TACTGAACCA ACTCATGAGT GGTCAGTGTC 15501 AAAAGCATCT TACGGATGGC ATGACAGTAA GAGAATTATG CAGTGCTGCC TTTTCGTAGA ATGCCTACCG TACTGTCATT CTCTTAATAC GTCACGACGG 15551 ATAACCATGA GTGATAACAC TGCGGCCAAC TTACTTCTGA CAACGATCGG TATTGGTACT CACTATTGTG ACGCCGGTTG AATGAAGACT GTTGCTAGCC 15601 AGGACCGAAG GAGCTAACCG CTTTTTTGCA CAACATGGGG GATCATGTAA TCCTGGCTTC CTCGATTGGC GAAAAAACGT GTTGTACCCC CTAGTACATT 15651 CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC GAGCGGAACT AGCAACCCTT GGCCTCGACT TACTTCGGTA TGGTTTGCTG 15701 GAGCGTGACA CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT CTCGCACTGT GGTGCTACGG ACATCGTTAC CGTTGTTGCA ACGCGTTTGA 15751 ATTAACTGGC GAACTACTTA CTCTAGCTTC CCGGCAACAA TTAATAGACT TAATTGACCG CTTGATGAAT GAGATCGAAG GGCCGTTGTT AATTATCTGA 15801 GGATGGAGGC GGATAAAGTT GCAGGACCAC TTCTGCGCTC GGCCCTTCCG CCTACCTCCG CCTATTTCAA CGTCCTGGTG AAGACGCGAG CCGGGAAGGC 15851 GCTGGCTGGT TTATTGCTGA TAAATCTGGA GCCGGTGAGC GTGGGTCTCG CGACCGACCA AATAACGACT ATTTAGACCT CGGCCACTCG CACCCAGAGC 15901 CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC CGTATCGTAG GCCATAGTAA CGTCGTGACC CCGGTCTACC ATTCGGGAGG GCATAGCATC 15951 TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG AATAGATGTG CTGCCCCTCA GTCCGTTGAT ACCTACTTGC TTTATCTGTC 16001 ATCGCTGAGA TAGGTGCCTC ACTGATTAAG CATTGGTAAC TGTCAGACCA TAGCGACTCT ATCCACGGAG TGACTAATTC GTAACCATTG ACAGTCTGGT 16051 AGTTTACTCA TATATACTTT AGATTGATTT AAAACTTCAT TTTTAATTTA TCAAATGAGT ATATATGAAA TCTAACTAAA TTTTGAAGTA AAAATTAAAT 16101 AAAGGATCTA GGTGAAGATC CTTTTTGATA ATCTCATGAC CAAAATCCCT TTTCCTAGAT CCACTTCTAG GAAAAACTAT TAGAGTACTG GTTTTAGGGA 16151 TAACGTGAGT TTTCGTTCCA CTGAGCGTCA GACCCCGTAG AAAAGATCAA ATTGCACTCA AAAGCAAGGT GACTCGCAGT CTGGGGCATC TTTTCTAGTT 16201 AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA TCCTAGAAGA ACTCTAGGAA AAAAAGACGC GCATTAGACG ACGAACGTTT 16251 CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA GTTTTTTTGG TGGCGATGGT CGCCACCAAA CAAACGGCCT AGTTCTCGAT 16301 CCAACTCTTT TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA GGTTGAGAAA AAGGCTTCCA TTGACCGAAG TCGTCTCGCG TCTATGGTTT 16351 TACTGGTCTT CTAGTGTAGC CGTAGTTAGG CCACCACTTC AAGAACTCTG ATGACCAGAA GATCACATCG GCATCAATCC GGTGGTGAAG TTCTTGAGAC 16401 TAGCACCGCC TACATACCTC GCTCTGCTAA TCCTGTTACC AGTGGCTGCT ATCGTGGCGG ATGTATGGAG CGAGACGATT AGGACAATGG TCACCGACGA 16451 GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA GACGATAGTT CGGTCACCGC TATTCAGCAC AGAATGGCCC AACCTGAGTT CTGCTATCAA 16501 ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC TGGCCTATTC CGCGTCGCCA GCCCGACTTG CCCCCCAAGC ACGTGTGTCG 16551 CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG GGTCGAACCT CGCTTGCTGG ATGTGGCTTG ACTCTATGGA TGTCGCACTC 16601 CTATGAGAAA GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GATACTCTTT CGCCGTGCGA AGGGCTTCCC TCTTTCCGCC TGTCCATAGG 16651 GGTAAGCGGC AGGGTCGGAA CAGGAGAGCG CACGAGGGAG CTTCCAGGGG CCATTCGCCG TCCCAGCCTT GTCCTCTCGC GTGCTCCCTC GAAGGTCCCC 16701 GAAACGCCTG GTATCTTTAT AGTCCTGTCG GGTTTCGCCA CCTCTGACTT CTTTGCGGAC CATAGAAATA TCAGGACAGC CCAAAGCGGT GGAGACTGAA 16751 GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC TATGGAAAAA CTCGCAGCTA AAAACACTAC GAGCAGTCCC CCCGCCTCGG ATACCTTTTT 16801 CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG GCGGTCGTTG CGCCGGAAAA ATGCCAAGGA CCGGAAAACG ACCGGAAAAC 16851 CTCACATGTG GGAGGCTAGA GTACATTTAG GTGACACTAT AGAA GAGTGTACAC CCTCCGATCT CATGTAAATC CACTGTGATA TCTT

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

It is further to be understood that all values are approximate, and are provided for description.

Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes. 

1-32. (canceled)
 33. A Replication competent (RC) mut-4 Sindbis virus vector comprising SEQ. ID NOS.: 2-8.
 34. The RC mut-4 Sindbis virus vector of claim 33 further comprising a suicide gene.
 35. The RC mut-4 Sindbis virus vector of claim 34 wherein said suicide gene is herpes simplex virus thymidine kinase (HSVtk).
 36. The RC mut-4 Sindbis virus vector of claim 34 wherein said suicide gene is cytosine deaminase.
 37. The RC mut-4 Sindbis virus vector of claim 34 wherein said suicide gene is Varicella Zoster virus thymidine kinase.
 38. The RC mut-4 Sindbis virus vector of claim 34 wherein the genome of said vector is in a single component.
 39. (canceled)
 40. A RC mut-4 Sindbis virus vector comprising the proteins encoded by SEQ ID NOS: 2-5.
 41. The RC mut-4 Sindbis virus vector of claim 40 further comprising the proteins encoded by SEQ ID NOS: 6-7.
 42. The RC mut-4 Sindbis virus vector of claim 41 further comprising the proteins encoded by SEQ ID NO:
 8. 43. The RC mut-4 Sindbis virus vector of claim 42 further comprising a suicide gene.
 44. The RC mut-4 Sindbis virus vector of claim 43 wherein said suicide gene is HSVtk.
 45. The RC mut-4 Sindbis virus vector of claim 43 wherein said suicide gene is cytosine deaminase.
 46. The RC mut-4 Sindbis virus vector of claim 43 wherein said suicide gene is Varicella Zoster virus thymidine kinase.
 47. The RC mut-4 Sindbis virus vector of claim 43 wherein the genome of said vector is in a single component. 